METHOD AND CONSTRUCTS FOR DELIVERING DOUBLE STRANDED RNA tO PEST ORGANISMS

ABSTRACT

The present invention relates in generally to RNAi and its use in gene silencing. In particular, the present invention relates to methods and constructs for delivering double stranded RNA (dsRNA) to pest organisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application from U.S. non-provisionalapplication Ser. No. 11/666,017 which was a national stage filing under35 under 35 U.S.C. 371 of international application PCT/EP2005/011441,filed Oct. 25, 2005, which was published under PCT Article 21(2) inEnglish, and claims priority under 35 U.S.C. 119(e) to U.S. provisionalapplication 60/621,718, filed Oct. 25, 2004, and to U.S. provisionalapplication 60/621,718, filed Oct. 24, 2004 and to U.S. provisionalapplication 60/628,976, filed Nov. 18, 2004, the disclosures of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to RNAi and its use in genesilencing. Furthermore, the present invention relates to methods andconstructs for delivering double stranded RNA (dsRNA) to pest organisms.

BACKGROUND

Targeted inhibition of gene expression has been a long-felt need inbiotechnology and genetic engineering. In the last few years, advancesin nucleic acid chemistry and gene transfer have inspired new approachesto engineer specific interference with gene expression.

One of these approaches consists of double-stranded RNA inhibition(RNAi) as a tool for controlling gene expression, as described in WO99/32619 and WO 00/01846. Double-stranded RNA inhibition is based on theintroduction of RNA into a living cell to inhibit gene expression of atarget gene in that cell. The RNA has a region with double-strandedstructure. Double-stranded RNA (dsRNA) has the capability to rendergenes nonfunctional in a sequence-specific manner. When introduced intocells, dsRNA can activate mechanisms that target the degradation ofcognate cytoplasmic mRNAs and thus can effectively silence full geneexpression at the posttranscriptional level. RNAi has been observed inmany cell types from divergent eukaryotes, including protozoa, fungi,plants, invertebrates, and mammals. Once inside the cells, long dsRNAmolecules are cleaved into double-stranded small interfering RNAs(siRNAs) that are 21-25 base pairs in length by an enzyme withRNaseIII-like activity (Dicer). Cleavage into siRNAs is an early step inthe RNAi silencing mechanism. Introduction of double-stranded RNA(dsRNA) can elicit a gene-specific RNA interference response in avariety of organisms and cell types.

In plants this technology may be used for instance with the aim ofmodifying or improving plant resistance towards pathogens and pests. Thelatter technique may involve the uptake of the dsRNA by pest organismswhen feeding on the plants. In delivery by feeding, dsRNA may bedistributed to cells from the gut of the feeding organism in the samemanner as nutrients. It is also conceivable that dsRNA residing in“infected” cells could undergo successive rounds of cellular exit andre-entry into adjacent “uninfected” cells.

However, delivery of dsRNA to pest organisms by feeding has limits.Difficulties related to the delivery of dsRNA to feeding targetorganisms are numerous and may for instance involve the need to use veryhigh amounts of dsRNA in order to be effective. Also, dsRNA may easilybreak down in the plants or during delivery to the target organism.Furthermore, in order to be effective, the dsRNA molecules shouldefficiently be taken up by the pest and delivered to the correcttargeting site in the pest organisms.

Since the advent of double-stranded RNA inhibition there has beenrecognized a need for specialized constructs designed for site-directeddelivery of double-stranded RNA in a pest organism. While there arevarious methods available for directly and indirectly introducing dsRNAinto cells, it is clear that these methods are generally inefficient,and have practical limitation. Therefore, in view of the foregoing,there exists a need to develop tools and methods for the more efficientdelivery of dsRNA into pest cells for the purpose of achieving RNAi andto kill or paralyze the pest. The present invention aims to provideimproved methods and constructs useful in the delivery ofdouble-stranded RNA in pest organisms, including nematodes, insects andfungi. An object of the present invention is thus to provide dsRNAconstructs with improved properties to be effectively taken up in thecells or tissues of the pest species.

Insect, fungal and nematode pests are a major cause of damage to theworld's commercially important agricultural crops. Current strategiesaimed at reducing crop losses rely primarily on chemical pesticides.Alternatively transgenic crops with intrinsic pest resistance offer apromising alternative and continue to be developed. Pest-resistantplants can reduce pest population growth, the number of pesticideapplications and the environmental impact of pesticides. There remains agreat need in the art for plants showing resistance to pest organisms.Another object of the present invention is therefore to provide pestresistant plants showing resistance to pest organisms such as nematodes,insects and fungi.

SUMMARY

The present invention provides delivery molecules for facilitating thedelivery of a double-stranded ribonucleic acid molecule to a pestorganism, as well as various uses of the delivery molecule. The presentinvention also provides complexes of the delivery molecule and chimericRNAi molecules, i.e. RNA molecules comprising double stranded RNA forthe purpose of RNA interference.

Specifically, in a first aspect the present invention relates to an RNAdelivery molecule consisting of a polypeptide sequence comprising (i) atleast one RNA-binding domain, (ii) at least one targeting polypeptideable to bind to a cellular endocytosis and/or transcytosis receptormolecule and (iii) optionally at least one peptide linker and/or atleast one purification tag.

The advantages of the present invention include: the ease of introducingdouble-stranded RNA into cells or tissues, the low concentration of RNAwhich can be used, the stability of double-stranded RNA, and theeffectiveness of the inhibition. The present invention allows for theintroduction of a double-stranded RNA molecule, such as a smallinterfering RNA, into a cell of a pest organism with greater ease andefficiency than previously possible using conventional methods known inthe art. Accordingly, the RNA delivery molecule of the present inventionprovides a powerful tool for various agronomic and research applicationsrequiring the delivery of dsRNA into a target pest organism.

In another embodiment, the invention relates to a chimeric RNAi moleculecomprising

-   -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, and,    -   at least one nucleotide sequence corresponding to a target        nucleotide sequence of a target gene of a pest species.

The invention further also relates to a complex comprising an RNAdelivery molecule as defined herein and a chimeric RNAi molecule asdefined herein.

The present invention further provides various methods of using an RNAdelivery molecule described herein, including methods of facilitatingdelivery of a double-stranded ribonucleic acid molecule into a pestorganism. In a second aspect, the present invention therefore relates toa method for delivering a dsRNA molecule to a pest species, comprising:

-   -   co-expressing in a plant cell of:    -   (a) an RNA delivery molecule according to the present invention,        and    -   (b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene of the pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a) to        induce RNAi, and    -   feeding said plant cell to said pest species, and thereby        increasing the resistance of the plant (cell) towards the        feeding pest.

The present invention also relates to methods for down-regulatingexpression of a target gene in a pest species. In an embodiment suchmethod for down-regulating expression of a target gene in a pest speciescomprises:

-   -   co-expressing in a plant cell of:    -   a) an RNA delivery molecule according to the present invention,        and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene to be        down-regulated, and which further comprises a nucleotide        sequence which binds to the RNA-binding domain of the RNA        delivery molecule of (a), and    -   feeding said plant cell to said pest species, and thereby        increasing the resistance of the plant (cell) towards the        feeding pest.

In an embodiment, the chimeric RNAi molecule consists of an RNA moleculewhich comprises double-stranded RNA comprising annealed complementarystrands, one of which has a nucleotide sequence which is complementaryto at least part of a target nucleotide sequence of the target gene, andwhich further comprises a nucleotide sequence which binds to theRNA-binding domain of the RNA delivery molecule.

In another embodiment, the invention provides a method fordown-regulating expression of a target gene in a pest species whichcomprises:

-   -   co-expressing in a plant cell of a complex comprising an RNA        delivery molecule as defined herein and a chimeric RNAi molecule        comprising of at least one nucleotide sequence recognized by an        RNA-binding protein or RNA-binding domain of said RNA delivery        molecule and, at least one nucleotide sequence is complementary        to at least part of a target nucleotide sequence of a target        gene of a pest species, and    -   feeding said plant cell to said pest species, and thereby        increasing the resistance of the plant (cell) towards the        feeding pest.

Another aspect of the invention relates to methods for producing atransgenic plant that is resistant to a pest species, comprising:

-   -   co-expressing in a plant cell of:    -   a) an RNA delivery molecule according to the present invention,        and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene of said pest        species, and which further comprises a nucleotide sequence which        binds to the RNA-binding domain of the RNA delivery molecule of        (a), and a    -   regenerating a plant from said plant cell.

In another embodiment, the invention provides a method for producing atransgenic plant that is resistant to a pest species, comprising:

-   -   co-expressing in a plant cell of a complex comprising an RNA        delivery molecule as defined herein and a chimeric RNAi molecule        comprising of at least one nucleotide sequence recognized by an        RNA-binding protein or RNA-binding domain of said RNA delivery        molecule and, at least one nucleotide sequence corresponding to        a target nucleotide sequence of a target gene of a pest species,        and

regenerating a plant from said plant cell.

In yet another aspect the present invention also relates to the use ofan RNA delivery molecule according to the present invention for deliveryof a dsRNA molecule to a pest species and in particular to a targetsequence in a pest species.

In yet another aspect the present invention also relates to the use ofan RNA delivery molecule according to the present invention forproducing a transgenic plant.

In another aspect the present invention also relates to the use of acomplex comprising an RNA delivery molecule as defined herein and achimeric RNAi molecule as defined herein for down-regulating expressionof a target gene in a pest species.

In yet another aspect the present invention further relates to the useof a complex comprising an RNA delivery molecule as defined herein and achimeric RNAi molecule as defined herein for producing a transgenicplant, and for producing a transgenic plant that is resistant to a pestspecies.

In another aspect, the present invention further relates to a nucleicacid encoding an RNA delivery molecule of the invention and to a vectorcomprising said nucleic acid.

In another aspect, the present invention provides a host cell whichcomprises a nucleic acid encoding an RNA delivery molecule of theinvention or a vector comprising said nucleic acid.

In another aspect, the present invention relates to a kit comprising atleast one RNA delivery molecule as defined herein.

In another aspect, the invention relates to a composition comprising atleast one RNA delivery molecule as defined herein. Preferably, theinvention also relates to a composition comprising

-   -   a) at least one RNA delivery molecule as defined herein, and    -   b) at least one RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene of a pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a).

Preferably, the composition or kit according to the invention furthercomprise at least one excipient, e.g. an excipient which is suitable forkeeping the RNA delivery molecule or the composition in a stabilecondition.

The present invention further relates in another aspect to a transgenicplant resistant to a pest species, an essential derived variety thereof,plant part, plant cell or protoplast thereof obtainable by methodsaccording to the present invention.

The invention also provides a transgenic plant, essentially derivedvariety thereof, plant part, plant cell or protoplast thereof whichcomprises a nucleic acid encoding an RNA delivery molecule as definedherein, wherein said nucleic acid is heterologous to the genome of saidtransgenic plant, or an essentially derived variety thereof, plant part,plant cell or plant protoplast thereof.

The invention also provides a transgenic plant which comprises a vectorcomprising a nucleic acid encoding an RNA delivery molecule of theinvention.

The invention further relates to a plant, essentially derived varietythereof, plant part, plant cell or protoplast thereof wherein the plant,essentially derived variety thereof, plant part, plant cell orprotoplast thereof has been transformed with a nucleic acid encoding anRNA delivery molecule as defined herein.

In another aspect, the invention relates to a plant, essentially derivedvariety thereof, plant part, plant cell or protoplast thereof whichco-expresses:

-   -   a) an RNA delivery molecule according to the present invention,        and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene in a pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a).

In further embodiments, the present invention relates to progeny orparts or derivatives of plants obtained from a plant or essentiallyderived variety thereof according to the present invention.

In yet another aspect the present invention also relates to a chimericRNAi molecule comprising a nucleotide sequence recognized by anRNA-binding protein or RNA-binding domain, and a nucleotide sequencecorresponding to a target nucleotide sequence of a target gene of a pestspecies.

Additional aspects of the present invention will be apparent in view ofthe detailed description, which follows.

DESCRIPTION OF THE FIGURES

FIGS. 1 to 14 illustrate the nucleotide and amino acid sequences of RNAbinding polypeptides according to the present invention.

FIGS. 15 to 40 illustrate RNA sequences binding to the respective RNAbinding polypeptides according to the present invention.

FIG. 41 illustrates the consensus secondary structure in the Bacillussubtilis ribonucleic anti-terminator (RAT) reacting with the LicTRNA-binding domain.

FIG. 42 illustrates the consensus secondary structure of RNA stem-loopsthat bind to bacteriophage MS2 coat protein.

FIGS. 43 to 54 and 56 to 58 and 60 to 74 and 76 to 90 illustrate severalexamples of chimeric RNAi molecules according to the present invention(SEQ ID NOs 41 to 52 and SEQ ID NOs 54 to 56 and SEQ ID NOs 57 to 71 andSEQ ID NOs 72 to 86).

FIG. 55 is a schematic representation of a a chimeric RNAi moleculeaccording to the present invention, as further represented in any of SEQID NOs 41 to 52 and any of SEQ ID NOs 54 to 56.

FIG. 59 is another schematic representation of a chimeric RNAi moleculeaccording to the present invention, as further represented in any of SEQID NOs 57 to 71.

FIG. 75 is yet another schematic representation of a chimeric RNAimolecule according to the present invention, as further represented inany of SEQ ID NOs 72 to 86.

FIG. 91 represents the amino acid sequence of an RNA binding polypeptideaccording to the present invention, in particular of the N-terminaldomain of bacteriophage λ N protein comprising amino acids 1 to 36 (SEQID NO 87).

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes an RNA delivery molecule forfacilitating the delivery of double-stranded RNA into a target organism,as well as various uses thereof. Specifically, it has been found thatRNA delivery molecule may be bound to a double-stranded ribonucleic acidmolecule by means of linkage to an RNA binding domain to form a complexbetween a protein moiety, e.g. the RNA delivery molecule, and a nucleicacid moiety, e.g. an RNA molecule comprising the double stranded RNAthat causes interference. Such RNA delivery molecule greatly facilitatesuptake efficiency and allows for the efficient in vivo delivery of dsRNAinto cells, or tissues of target pest organisms.

While the present invention is primarily directed to the delivery of adouble-stranded ribonucleic acid molecule into a pest organism for thepurposes of RNA interference, the RNA delivery molecules describedherein may also be used to facilitate the delivery of other non-codingRNAs, such as small temporal RNAs, small nuclear RNAs, small nucleolarRNAs or microRNAs, which may be used in applications other than RNAinterference.

A. RNA Delivery Molecule

In accordance with the present invention, a delivery promoting moleculeis used to facilitate the uptake and the correct delivery of doublestranded RNA to a suitable target site in a plant-feeding pest organismfor the purpose of RNA interference.

The terms “RNA delivery module”, “RNA delivery molecule” and “RNAdelivery vehicle” are used herein as synonym and refer to themultidomain or multimodular protein which binds to an RNAi mediatingmolecule.

The term “RNAi” generally means “RNA interference”.

In one embodiment of the present invention, the RNA delivery moleculeconsists of a polypeptide sequence which comprises:

-   -   i) at least one RNA binding domain,    -   ii) at least one targeting polypeptide able to be endocytosed        and/or transcytosed or able to bind to a cellular endocytosis        and/or transcytosis receptor molecule    -   iii) optionally at least one peptide linker for linking the RNA        binding domain to the targeting polypeptide, and    -   iv) optionally a module comprising a purification tag.

The expression “at least one” in the context of the present inventionmeans at least two, at least three, at least four, at least five, atleast six, etc. and up to at least 10 or at least 15.

These modules can be swapped spatially. For example, the RNA bindingdomain can be at the N-terminus of the targeting polypeptide or can beat the C-terminus of the targeting polypeptide. The purification modulecan be at the N-terminus or at the C-terminus of the RNA deliverymolecule. Not all modules need to be present. For example, for proteinisolation the fourth module is included whereas for in vivo expressionit can be present or absent. The separate modules comprised in the RNAdelivery molecule will be discussed into more detail hereunder.

In a specific embodiment the invention relates to an RNA deliverymolecule consisting of a polypeptide sequence comprising (i) at leastone RNA-binding domain, (ii) at least one targeting polypeptide able tobind to a cellular endocytosis and/or transcytosis receptor molecule and(iii) optionally at least one peptide linker.

RNA-Binding Module

More in particular, a first module in the RNA delivery moleculeaccording to the present invention comprises an RNA binding module. Thismodule permits to specifically bind RNA to the RNA delivery molecule.The terms “RNA binding module”, “RNA binding molecule” and “RNA bindingprotein” are used herein as synonym and refer to a protein or anessential part, or a homologue thereof, which is capable of binding RNA.The RNA binding molecule comprises an “RNA binding domain”. An “RNAbinding domain” as used herein may bind double-stranded RNA genericallyor specifically, single-stranded RNA generically or specifically. TheRNA binding molecule may bind dsRNA, ssRNA structure specifically.

The term “essential parts thereof” or “functional part thereof” in thiscontext refers to parts of RNA binding proteins which are capable ofbinding dsRNA, ssRNA or which recognize and bind a secondary structurein an RNA.

The term “homologue” of an RNA binding protein as used herein refers toa protein which has an amino acid sequence that has at least 30%identity, preferably at least 40%, 50%, 60%, 70%, 80% or 90% identity,most preferably at least 95% identity with a functional portion of theamino acid sequence of an RNA binding protein. It should be understoodthat instead of % “identity”, also the corresponding % “similarity” canbe used to define homologues according to the invention.

The term “functional portion” in the context of the present inventionmeans a portion or fragment of the RNA binding protein comprising theRNA-binding domain.

As used herein, the “% identity and % similarity” are calculated usingmatcher (EMBOSS, based on Bill Pearson's ‘lalign’ application, version2.0u4 Feb. 1996). Lalign is based on an algorithm developed by X. Huangand W. Miller (Adv. Appl. Math. (1991) 12:337-357) for the “sim”program, which is a linear-space version of an algorithm described by M.S. Waterman and M. Eggert (J. Mol. Biol. (1987) 197:723-728). AEBLOSUM62 was used as a substitution matrix, and a gap penalty of 14 andan extended penalty of 4 for the alignment.

For the purpose of the present invention, the RNA binding protein or RNAbinding domain binds an RNA molecule comprising the double-stranded RNAcausing interference in a pest species. The RNA binding domain may binddirectly to said double-stranded RNA causing interference, throughrecognition of and binding to one or more RNA sequences within saiddouble-stranded RNA, or through recognition of and binding to an RNAstructure within said double-stranded RNA. Alternatively, the RNAmolecule comprising the double-stranded RNA causing interferencecomprises a further RNA sequence which is recognized by the RNA bindingprotein or RNA binding domain as being a binding site specific for thesaid RNA binding protein or domain. Said further RNA sequence may bedouble-stranded, single-stranded or may comprise an RNA structure whichis specifically recognized and bound by the RNA binding protein or RNAbinding domain.

It should be noted that each of the RNA binding proteins or RNA bindingdomains may bind to specific RNA sequences, patterns, recognition sites,RNA structures or the like.

Preferred examples of RNA binding proteins include but are not limitedto coliphage HK022 Nun protein, Bacillus subtilis LicT protein, orbacteriophage MS2 coat protein or essential parts, or homologuesthereof.

In a preferred embodiment, the RNA binding module comprises thecoliphage HK022 Nun protein (Genbank entries: nucleotide sequence:X16093 (whole viral sequence); amino acid sequence: P18683, VNBPHK) oressential parts thereof. The nucleotide and amino acids sequencescorresponding to the gene encoding the coliphage HK022 Nun protein areillustrated in FIGS. 1 (SEQ ID NO 1) and 2 (SEQ ID NO 2), respectively.The HK022 Nun of phage HK022 is a transcription termination factor thatacts highly template and site specific. Nun terminates transcriptionuniquely on phage lambda templates, thereby in competition with lambda Nprotein for the common binding site, the nut boxB RNA. This is a 15-merRNA hairpin containing a purine-rich pentaloop. Nun protein, as the Nprotein, belongs to a family of arginine-rich motif binding proteins.They bind to the major groove of the boxB RNA, which adopts a typicalhairpin confirmation closed by an apical tetraloop (Faber et al., J.Biol. Chem., 2001, 276, 32064 and reference therein). The protein is analpha helix connecting to the hairpin of the RNA. There is a high andspecific affinity of the RNA binding domain in the Nun protein towardsthe boxbRNA. The peptide structure seems highly charged while the RNA isrich in purines. Both are quite special entities.

In another preferred embodiment the RNA binding module comprisesessential parts (for instance comprising any of SEQ ID Nos 4, 6 or 8) ofthe coliphage HK022 Nun protein.

Preferred examples hereof comprise but are not limited to;

-   -   the N-terminal domain of coliphage HK022 Nun protein 1 to 47        (SEQ ID NO 4) comprising the RNA-binding domain,    -   the N-terminal domain of coliphage HK022 Nun protein 13 to 47        (SEQ ID NO 6) comprising the RNA-binding domain,    -   the N-terminal domain of coliphage HK022 Nun protein 22 to 47        (SEQ ID NO 8) comprising the RNA-binding domain,

In yet another preferred embodiment the RNA binding module comprises

-   -   proteins belonging to the same arginine-rich motif (ARM) family        as coliphage HK022 Nun protein such as for instance        bacteriophage λ N protein, or    -   RNA binding domains of proteins belonging to the same        arginine-rich motif (ARM) family as coliphage HK022 Nun such as        for instance the N-terminal domain of bacteriophage λ N protein        comprising amino acids 1 to 36 (as represented in SEQ ID NO 87)        or amino acids 1 to 22.

In another preferred embodiment the RNA binding module compriseshomologues of the coliphage HK022 Nun protein. Therefore, according to afurther embodiment, the invention also relates to an RNA deliverymolecule as described above, wherein said at least one RNA-bindingdomain comprises a polypeptide having at least 30% identity, preferablyat least 40%, 50%, 60%, 70%, 80% or 90% identity, most preferably atleast 95% identity with the coliphage HK022 Nun protein (or with thepolypeptide as represented in SEQ ID NO 2). It should be understood thatinstead of % “identity”, also the corresponding % “similarity” can beused to define homologues according to the invention.

In a preferred example, the N-terminal binding-domain of thebacteriophage HK022 Nun protein (SEQ ID NO 2) and in particular thepeptide with sequence SEQ ID NO 8 interacts with the box B RNA sequence(SEQ ID NO 15) and in particular with the 11mer RNA sequence representedin SEQ ID NO 16. (Scharpf et al., Eur. J. Biochem. 267, p2397 (2000),Faber et al., J. Biol. Chem. 276, 34, p32064 (2001)).

In another embodiment, the RNA binding module comprises the Bacillussubtilis LicT protein or essential parts thereof (Genbank/Swissprot/PIRentries: nucleotide sequence: Z28340 (D83026, Z99124 for larger genomicfragments); amino acid sequence: P39805, S47216, BAA11696, CAA82194).The nucleotide and amino acid sequences corresponding to the geneencoding the subtilis LicT protein are illustrated in FIGS. 9 (SEQ ID NO9) and 10 (SEQ ID NO 10), respectively. The transcriptionalanti-terminator LicT from Bacillus subtilis is a regulatory protein ableto prevent the premature arrest of transcription. When activated, LicTbinds to RNA of around 30 nucleotides. This RNA adopts a hairpinconformation containing a variable apical loop and two asymmetricinternal loops. LicT belongs to a group of antiterminator proteins(involved in carbohydrate metabolism control in Gram-positive andGram-negative bacteria) for which the RNA recognition is embedded in theN-terminal protein fragment. This domain binds as a beta-strandedsymmetric dimer that shares no structural homology with other knownRNA-binding motifs (EMBO; 2002, 21, 1987 and references therein). Thereis a high level of sequence identity both in the protein family as inthe RNA sequence for the AA/nucleotides that interact with each other.Most of the conserved residues of the protein are in direct contact(hydrophobic or via Hbond donor/acceptor) with the RNA. Those which arenot in contact do not seem to be important for RNA binding, but mightplay a role for intra-inter protein interactions during theantitermination process. Nine AA (from position 5 till 31) interact withthe RNA, all of them highly conserved. Six of them are crucial for thein vivo anti-terminal activity or in vitro RNA binding.

In another preferred embodiment the RNA binding module comprisesessential parts (for instance comprising SEQ ID NO 12) of the Bacillussubtilis LicT protein. Preferred examples hereof comprise but are notlimited to the N-terminal domain of Bacillus subtilis LicT proteincomprising amino acids 1 to 56 (SEQ ID NO 12) containing the RNA-bindingdomain.

In yet another preferred embodiment the RNA binding module comprises

-   -   proteins belonging to the same anti-terminator (AT) family as        Bacillus subtilus LicT protein such as for instance the Bacillus        subtilis SacY protein or the Escherichia coli BgIG protein,    -   the RNA binding domain of proteins belonging to the same        anti-terminator (AT) family as Bacillus subtilus LicT protein        such as for instance N-terminal domain of Bacillus subtilis SacY        protein comprising amino acids 1 to 55 of the SacY protein or        the N-terminal domain of Escherichia coli BgIG protein        comprising amino acids 4 to 60 of the BgIG protein.

In another preferred embodiment the RNA binding module compriseshomologues of the Bacillus subtilis LicT protein. Therefore, accordingto a further embodiment, the invention also relates to an RNA deliverymolecule as described above, wherein said at least one RNA-bindingdomain comprises a polypeptide having at least 30% identity, preferablyat least 40%, 50%, 60%, 70%, 80% or 90% identity, most preferably atleast 95% identity with the Bacillus subtilis LicT protein (or with thepolypeptide as represented in SEQ ID NO 10), preferably said RNA-bindingdomain comprises a polypeptide having at least 60% identity, preferablyat least 70%, 80% or 90% identity, most preferably at least 95% identitywith the RNA binding domain of the Bacillus subtilis LicT protein (i.e.with the polypeptide as represented in SEQ ID NO 12). A non-exhaustivelist of such homologous polypeptides is given in Table 1, and mayfurther include representative members of the LicT/SacY family oftranscriptional ATs from Bacillus subtilis, Bacillus stearothermophilus,Listeria mnocytogenes, Clostridium longisporum, Clostridiumacetobutylicum, Staphylococcus carnosus, Streptococcus agalactiae,Lactococcus lactis, Lactobacillus casei, E. coli and Erwiniachrysanthemi. It should be understood that instead of % “identity”, alsothe corresponding % “similarity” can be used to define homologuesaccording to the invention.

TABLE 1 Homologues of the Bacillus subtilis LicT protein Genbankaccession accession Homologues Identity * Similarity * number number **Antiterminator 29.7 53.6 2632149 CAA74358 [Staphylococcus cornosus]lact2 31.4 53.6 6016464 P24401 beta-glucoside operon antitermintator54.2 76.6 114108 P26211 cryptic beta-glucoside bgl operon antiterminator42.4 69.9 114961 P11989 hypothetical protein 40.4 66.7 2765188 CAA72900[Streptococcus agalactiae] Levansucrase and sucrase synthesis operon37.4 64 134180 P15401 antiterminator transcription antiterminator 38.665.7 1737500 AAB38977 [Geobacillus stearothermophilus] SacPA operonantiterminator 41.2 67.2 134178 P26212 transcription antiterminator;BgIR 37.9 62.5 4704716 AAD28229 [Enterococcus faecium] regulatoryprotein 47.1 69.7 7304840 AAC05712 [Clostridium longisporum]Antiterminator 35.3 62.2 4138148 CAA07716 [Listeria monocytogenes] BgIR41.5 63.2 551875 AAA57135 transcription antiterminator 31.4 60.6 2154723CAA72077 [Bacillus subtilis] antiterminator homolog 33.1 56.1 2197103AAB61291 [Escherichia coli] * identity or similarity with theRNA-binding protein LicT. ** accession number according to otherdatabases such as Swissprot or PIR

In a preferred embodiment, the LicT RNA-binding domain (comprising SEQID NO 12) interacts with the Bacillus subtilis ribonucleicantiterminator (RAT) that has a secondary structure as depicted in FIG.41 or binds to an RNA having a sequence as given in any of SEQ ID NOs 17to 34 (Scharpf, Sticht et al., Eur. J. Biochem. 267, p2397 (2000),Scharpf et al. 2002, Yang 2000). Any of SEQ ID NOs 17 to 33 correspondswith the RNA binding recognition site in the homologous protein in therepresentative members of the LicT/SacY family of transcriptional Atsfrom Bacillus subtilis, Bacillus stearothermophilus, Listeriamnocytogenes, Clostridium longisporum, Clostridium acetobutylicum,Staphylococcus carnosus, Streptococcus agalactiae, Lactococcus lactis,Lactobacillus casei, E. coli and Erwinia chrysanthemi. The 29-mersequence recognition pattern for binding to LicT homologues isrepresented in FIG. 34 (SEQ ID NO: 34).

In yet another preferred embodiment, the RNA binding module comprisesthe bacteriophage MS2 coat protein or essential parts thereof(Genbank/Swisprot/PIR entries: nucleotide sequence: V00642 (wholegenome), NC_(—)001417 (whole genome); amino acid sequence: PO₃₆₁₂,CAA23989, NP_(—)040648, VCBPM2, 721932A). FIG. 13 (SEQ ID NO 13) and 14(SEQ ID NO 14) illustrate the nucleotide and amino acid sequences of thegene encoding bacteriophage MS2 coat protein. A dimer of the MS2 coatprotein binds specifically to a 19 nt long hairpin in the singlestranded viral RNA genome which results in the repression of thereplicase translation. The complex also functions as an initiation sitefor viral assembly and controls the encapsidation of the cognate RNA invivo (Nucleic Acid Research, 2002, 30, 2678-2685, Nature, 1994, 13,623-626 and references therein).

In another preferred embodiment the RNA binding module comprisesessential parts (comprising the RNA binding domain) of the bacteriophageMS2 coat protein. Other preferred examples of suitable RNA bindingmodules according to the present invention may comprise but are notlimited to

-   -   coat proteins of other Leviviridae such as those of subgroup I,        e.g. f2, R17, or subgroup II, e.g. JP34,    -   RNA-binding domains of coat proteins of other Leviviridae        (subgroup I, e.g. f2, R17, or subgroup II, e.g. JP34).

In another preferred embodiment the RNA binding module compriseshomologues of the bacteriophage MS2 coat protein. Therefore, accordingto a further embodiment, the invention also relates to an RNA deliverymolecule as described above, wherein said at least one RNA-bindingdomain comprises a polypeptide having at least 30% identity, preferablyat least 40%, 50%, 60%, 70%, 80% or 90% identity, most preferably atleast 95% identity with the bacteriophage MS2 coat protein (or with thepolypeptide as represented in SEQ ID NO 14), preferably said RNA-bindingdomain comprises a polypeptide having at least 60% identity, preferablyat least 70%, 80% or 90% identity, most preferably at least 95% identitywith the RNA binding domain of the MS2 coat protein. A non-exhaustivelist of such homologous polypeptides is given in Table 2. It should beunderstood that instead of % “identity”, also the corresponding %“similarity” could be used to define homologues according to theinvention.

TABLE 2 Homologues of the bacteriophage MS2 coat protein accessionHomologous Identity * Similarity * number ** Coat protein bacteriophageR17 98 100 PO3613 Coat protein enterobacterio phage M12 98 98.4 Q9T1C7Coat protein bacteriophage JP501 95 97.7 Q9MCD7 Coat proteinbacteriophage Fr 87 91.5 P03614 Coat protein bacteriophage Ku1 63 79.7Q9MBL2 Coat protein bacteriophage JP34 65 78.9 P34700 Coat proteinbacteriophage GA 63 77.2 P07234 Coat protein enterobacterio phage MX1 2448.6 O64307 Coat protein bacteriophage PRR1 30 45.4 P03616 Coat proteinbacteriophage SP 23 44.6 P09673 Coat protein bacteriophage M11 26 43.6O64303 Coat protein bacteriophage Q-beta 23 43.3 P03615 Coat proteinenterobacteriol phage NL95 25 42.4 O64310 * identity or similarity withthe MS2 coat protein. ** accession number according to databases such asGenbank/Swissprot/PIR

In a preferred embodiment, the bacteriophage MS2 coat protein (SEQ ID NO14) interacts with RNA stem-loops that have a secondary structure asdepicted in FIG. 42 or binds to RNA sequences having a sequence as givenin any of SEQ ID NOs 35 to 40 (Rowsell et al., Nat. Struct. Biol. 5, 11,p970, 1998). The sequence recognition pattern comprises:5′-N₁N₂N₃N₄N₅PuN₇N₉AN₁₀PyAN₁₃N₁₄N₁₅N₁₆N₁₇N₁₈N₁₉-3′ (SEQ ID NO 40); inthis formula, N₁-N₅ are the reverse complementary of N₁₅-N₁₉ and N₇-N₈are the reverse complementary of N₁₃-N₁₄, and preferably with therestriction that N₆ (Pu) does not pair with N₁₄.

The present invention thus relates to an RNA delivery molecule asdescribed above, wherein said at least one RNA-binding domain (i) ischosen from:

-   -   a polypeptide comprising the coliphage HK022 Nun protein (for        instance as represented in SEQ ID NO 2), a homologue thereof, or        a fragment thereof comprising the RNA-binding domain, said        fragment preferably comprising amino acids 1 to 47 (SEQ ID NO 4)        of the aminoterminal sequence, more preferably comprising amino        acids 13 to 47 (SEQ ID NO 6) of the aminoterminal sequence, more        preferably comprising amino acids 22 to 47 (SEQ ID NO 8) of the        aminoterminal sequence,    -   a polypeptide comprising the Bacillus subtilis LicT protein (for        instance as represented in SEQ ID NO 10), a homologue thereof,        or a fragment thereof comprising the RNA-binding domain, said        fragment preferably comprising amino acids 1 to 56 (SEQ ID        NO 12) of the aminoterminal sequence, and    -   a polypeptide comprising the bacteriophage MS2 coat protein (for        instance as represented in SEQ ID NO 14), a homologue thereof,        or a fragment thereof comprising the RNA-binding domain.

Targeting Module

A second module of the RNA delivery molecule comprises a targetingmodule. The terms “targeting module”, “targeting molecule”, “targetingprotein” and “targeting polypeptide” are used herein as synonym andrefer to a protein, or an essential part, or a homologue thereof capableof targeting the RNA delivery molecule to a targeting site in a livingpest organism. The term “essential parts thereof” refers to parts of atargeting protein which are capable of targeting the RNA deliverymolecule to a targeting site in a living pest organism. The term“homologue” of a targeting protein as used herein refers to a proteinwhich has an amino acid sequence that has at least 30% identity,preferably at least 40%, 50%, 60%, 70%, 80% or 90% identity, mostpreferably at least 95% identity to a functional portion of the aminoacid sequence of a targeting protein. It should be understood thatinstead of % “identity”, also the corresponding % “similarity” could beused to define homologues according to the invention.

The term “functional portion” in the context of the present inventionmeans a portion or fragment of the targeting protein comprising thedomain or sequence which binds to the target cell or tissue in the pestorganism.

As used herein the term “targeting site” refers to a specific cell ortissue in a living pest organism, as defined herein, to which the RNAdelivery molecule according to the present invention is targeted.

The targeting module preferably comprises a protein which is capable ofbeing endocytosed and/or transcytosed in a cell of the pest organism, ora protein able to bind an endocytosis and/or transcytosis receptormolecule present on a cell or a tissue of the pest organism, or anycombinations thereof. In a preferred embodiment, the invention relatesto an RNA delivery molecule as described above, wherein said at leastone targeting polypeptide binds to a gut cell endocytosis ortranscytosis receptor molecule. In another preferred embodiment, theinvention relates to an RNA delivery molecule as described above,wherein said at least one targeting polypeptide binds to a tissue cellendocytosis or transcytosis receptor molecule.

“Endocytosis” is defined herein as the cellular uptake of macromoleculesand particulate substances by localized regions of the plasma membranethat surround the substance and pinch off to form an intracellularvesicle. “Receptor-mediated endocytosis” is an essential process in alleukaryotes, including invertebrates such as insects or nematodes, and isrequired for general cellular functions, including uptake of nutrients(e.g., low-density lipoprotein [LDL] or transferrin) and recycling ofmembranes and membrane proteins.

“Transcytosis” is defined herein as the process by which a molecule mayenter through one side of a cell and then migrate across the cell toexit on the other side. Transcytosis refers to the transport ofsubstances across an epithelium by uptake into and release from coatedvesicles. Also “receptor-mediated transcytosis” is an essential processin eukaryotes.

In certain embodiments of the present invention, the RNA deliverymolecule comprises one targeting molecule, for instance a targetingmolecule which allows endocytosis into the gut cell of a pest organism.In another example, the targeting molecule allows transcytosis from thelumen of the gut to the coelumic fluid or haemolymph of the pestorganism. In other embodiments of the present invention the RNA deliverymolecule comprises one targeting molecule which allows endocytosis intoa tissue cell of the pest organism, such as for instance, but notlimited to, a muscle cell, a gonade cell, a nerve cell. In anotherexample, the targeting molecule allows transcytosis from an endothelialcell lining an organ to the lumen of said organ of the pest organism. Instill other embodiments of the present invention, the RNA deliverymolecule comprises two targeting molecules, for instance one targetingmolecule which allows transcytosis from the gut cell of a pest organismto the coelumic fluid or haemolymph of the pest organism, and anothertargeting molecule which allows endocytosis into a tissue cell of thepest organism.

In particular examples, where the pest species is a fungal species, theterm “endocytosis” is to be understood in the context of the fungi'sfeeding process. Upon feeding, fungi secrete enzymes or proteins in theprocess used by said fungal species for uptake of nutrients. As part ofthe feeding process, said enzyme or protein (bound or not to nutrients)is taken up again by said fungal species through endocytosis processes.Therefore, according to the present invention, the complex between theRNA delivery molecule and the dsRNA molecule that may cause RNAinterference in the fungus, may simultaneously be taken up by the fungusthrough binding of a domain in the RNA delivery molecule to a secretedenzyme or protein (or to a part thereof, which is re-uptaken). Thepresent invention is thus equally applicable to target the RNA deliverymolecule or the complex to endocytosed proteins (or to their receptorson the fungal cell wall) in fungi.

In another embodiment, the targeting molecule comprises at least onesequence which is recognized by a gut cell or a receptor on the gut cellof a pest organism, and which recognition triggers the endocytosis ortranscytosis of the complex between the RNA delivery molecule and thechimeric RNAi molecule.

Preferred examples of endocytosis receptor binding molecules suitablefor use in the present invention comprise but are not limited to

-   -   transferrin proteins from any pest species, such as nematode or        insect species such as for instance Caenorhabditis elegans,        Drosophila melanogaster, Meloidygyne incognita, Corn Root Worm,        Tabacco Budworm etc. . . . ;    -   serum transport proteins from any pest species, such as nematode        or insect species    -   homologues of the above mentioned proteins in fungal species    -   vitellogenin, low density lipoprotein, transcobalamin, yolk        proteins, and/or homologues thereof    -   Antibodies raised against receptors involved in        receptor-mediated endocytosis in the pest organism    -   Antibodies such as IgG, IgA, IgE, (Trf)-specific camel        antibodies    -   Antibodies raised against pest gut proteins, preferably camel        antibodies    -   lectins such as snowdrop lectin, concanavalin A, Ricin    -   coat proteins from viruses such as Luteoviridae,    -   coat proteins from viruses such as Rous sarcoma virus, Semliki        forest virus, Vesicular stomatitis virus, Adenovirus,    -   hormones and growth factors such as Insulin, Epidermal Growth        Factor, Growth Hormone, thyroid stimulating hormone, Nerve        Growth Factor, Calcitonin, Glucagon, Prolactin, Luteinizing        Hormone, Thyroid hormone, Platelet Derived Growth Factor,        Interferon, Catecholamines.

In a preferred embodiment, the endocytosis receptor binding moleculesuitable for use in the present invention comprises transferrin proteinsfrom any pest species, such as nematode or insect species, lectins suchas snowdrop lectin, and coat proteins from viruses such as Luteoviridae.

Preferred examples of transcytosis receptor binding molecules suitablefor use in the present invention comprise but are not limited to:

-   -   transferrin proteins from any pest species, such as nematode or        insect species such as for instance    -   lectins such as snowdrop lectin, concanavalin A, Ricin    -   coat proteins from viruses such as Luteoviridae,

In another preferred embodiment, the transcytosis receptor bindingmolecule suitable for use in the present invention comprise lectins suchas snowdrop lectin, and coat proteins from viruses such as Luteoviridae.

In the examples section, a non-limiting number of RNA delivery moleculesare described, comprising targeting polypeptides according to theinvention.

According to yet another aspect of the invention, the RNA deliverymolecule does not comprise a targeting polypeptide. The RNA deliverymolecules according to this aspect of the invention may further compriseall elements of RNA delivery molecules as described herein. The majoradvantage of these RNA delivery molecules is that it may protect RNAimolecules by binding them in the complex, i.e. protection from plantDicer and/or other plant degradation systems. Another advantage is thatthe RNA delivery molecule may direct and/or store the chimeric RNAimolecule of the invention in plant organelles, for instance in case thechimeric RNAi molecules do not encode such signals.

Linker Module

The RNA delivery module according to the invention preferably comprisesa third module which consists of at least one peptide linker for linkingthe first to the second module. Said linker polypeptide preferablycomprises a polypeptide of at least 3, preferably at least 4 or 5, mostpreferably at least 7, and more preferably at least 12 amino acids.Preferably said linker is a polypeptide comprising between 3 and 15amino acids. Preferably said linker is a polypeptide comprisingnon-charged amino acids such as glycine, serine, cysteine, asparagine,tyrosine, glutamine, alanine, valine, proline, threonine, and preferablyglycine or serine. A preferred example of a linker polypeptide comprisesthe polypeptide having the amino acid sequence GGGGSGGGGSGGGGS (SEQ IDNO 53). In preferred examples, the linker polypeptide comprises one ofthe following:

-   -   a factor Xa cleavage site, i.e. IEDR (SEQ ID NO 88),    -   a thrombin cleavage site, i.e. LVPGRS (SEQ ID NO 89)    -   PGISGGGGG (SEQ ID NO 90)    -   GGS, or    -   AAA.

In a preferred embodiment, the present invention thus relates to an RNAdelivery molecule as described above further comprising at least onelinker module, preferably a linker module as described above.

Purification Module

The RNA delivery module according to the invention may further comprisea fourth module which consists of a purification tag. Said purificationtags, defined by specific amino acid sequences, enables the easypurification of the tagged delivery molecule. Preferred examples ofpurification modules comprise but are not limited to His-tag, GST-tag,c-myc-tag, FLAG-tag, E-tag.

The invention thus further relates to an RNA delivery molecule asdescribed above, further comprising a purification tag, preferably apurification tag as described above.

B. Chimeric RNAi Molecule Chimeric RNAi Molecule

The term “chimeric RNAi molecule” or “RNAi molecule” as used hereinmeans a sequence that contains

-   -   a nucleotide sequence corresponding to the target nucleotide        sequence of the target gene of the pest organism and capable of        inducing RNA interference, and    -   an RNA sequence recognized by RNA-binding protein or RNA-binding        domain.

In the context of the present invention, the sequence corresponding tothe target nucleotide sequence is contained in a double-stranded RNA(dsRNA) wherein said double-stranded RNA comprises annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to at least part of a target nucleotide sequence of atarget gene. The expressions “chimeric RNAi molecule”, “RNAi mediatingmolecule”, and “chimeric RNAi mediating molecule” all relate to asequence containing a sequence corresponding to a target nucleotidesequence and the RNA sequence recognized by the RNA-binding protein orRNA-binding domain.

Preferred examples of chimeric RNAi molecules are molecules that consistof a target RNA sequence and an RNA sequence that is recognized by anRNA binding module as described in the present invention. The RNAsequences that are recognized by an RNA binding protein or RNA bindingdomain include but are not limited to RNA sequences recognized by thecoliphage HK022 Nun protein, Bacillus subtilis LicT protein, orbacteriophage MS2 coat protein or essential parts, or homologues of saidRNA binding proteins. The RNA sequences that are recognized by an RNAbinding module as described in the present invention are specific,meaning that RNA sequences identical or comprising the consensussequences as depicted in FIGS. 15 to 40 or as represented in FIGS. 41and 42 are not or rarely found in a host (plant), target species ornon-target species genome.

In a preferred embodiment, the present invention relates to a chimericRNAi molecule comprising

-   -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, for instance as represented in        any of SEQ ID NOs 15 to 40, or an RNA secondary structure as        represented in FIG. 41 or 42, and    -   at least one nucleotide sequence corresponding to a target        nucleotide sequence of a target gene of a pest species.

Table 3 provides an overview of different RNA sequences contained inchimeric RNAi molecules according to the present invention which arespecifically recognized by a Nun, LicT or MS2 RNA-binding protein orRNA-binding domain as indicated.

TABLE 3 Amino acid sequence Nucleic acid sequence SEQ ID NOs 2, 4, 6 or8 SEQ ID NO 15 and 16 (coliphage HK022 Nun protein (*)) SEQ ID NO 10 or12 SEQ ID NOs 17 to 34 (Bacillus subtilis LicT protein (*)) SEQ ID NO 14SEQ ID NOs 35 to 40 (bacteriophage MS2 coat protein (*)) (*) oressential parts, or homologues thereof

According to a preferred embodiment, said chimeric RNAi molecule is amolecule as described in Example 2. According to further embodiments,the chimeric RNAi molecule has a sequence as represented in any of FIGS.43 to 54 (any of SEQ ID NOs 41 to 52) or in any of FIGS. 56 to 58 (anyof SEQ ID NOs 54 to 56), or in any of FIGS. 60 to 74 (any of SEQ ID NOs57 to 71), or in any of FIGS. 76 to 90 (any of SEQ ID NOs 72 to 86).

In yet another embodiment, the present invention relates to a chimericRNAi molecule according to the present invention as represented by anyof SEQ ID NOs 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55 or56. In another embodiment, the present invention relates to a chimericRNAi molecule according to the present invention as represented by anyof SEQ ID NOs 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71. In yet another embodiment, the present invention relates to achimeric RNAi molecule according to the present invention as representedby any of SEQ ID NOs 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86.

Table 4 provides an overview of different examples of chimeric RNAimolecules according to the present invention which contain an RNAsequence that is specifically recognized by a Nun, LicT or MS2RNA-binding protein or RNA-binding domain or essential parts, orhomologues thereof as indicated.

TABLE 4 SEQ ID NOs 2, 4, 6 or 8 SEQ ID NOs 45, 46, 47, 48, 55, 61, 62,63, 64, 70, (coliphage HK022 Nun protein (*)) 76, 77, 78, 79, 85 SEQ IDNO 10 or 12 SEQ ID NOs 41, 42, 43, 44, 54, 57, 58, 59, 60, 69, (Bacillussubtilis LicT protein (*)) 72, 73, 74, 75, 84 SEQ ID NO 14 SEQ ID NOs49, 50, 51, 52, 56, 65, 66, 67, 68, 71, (bacteriophage MS2 coat protein(*)) 80, 81, 82, 83, 86 (*) or essential parts, or homologues thereof

In another embodiment, the present invention relates to the use of achimeric RNAi molecule as defined herein, and in particular asrepresented by any of SEQ ID NOs 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86 fordown-regulation of target genes in a pest species, for delivering dsRNAand/or for producing transgenic plants resistant to pest organisms.

Target Genes

A “target gene” as used herein means a gene that needs to be silenced inthe target (pest) species. The target gene may be selected from thegenome of any species as described herein. According to a preferredembodiment, the target sequence is chosen from the genome of an organismwhich organism is different from the organism in which the dsRNA capableof causing interference is expressed. This means that the dsRNA isexpressed in one (host) cell or organism and is subsequently transferred(or taken up by) to another cell or organism comprising the target gene.According to one specific embodiment of the present invention, the dsRNAis expressed in the plant or a plant cell and the target gene is chosenfrom the genome of a bacterium, a virus or an invertebrate, moreparticularly from a plant pest species such as a nematode, fungus or aninsect. In the present context, the expression “dsRNA” relates to doublestranded RNA capable of causing RNA interference. In another embodiment,the dsRNA is expressed in a bacterial or fungal cell and the bacterialor fungal cell is taken up or eaten by the pest species. According tostill another embodiment, the dsRNA is isolated from, or purified from,the bacterial or fungal cell expressing the dsRNA, and the dsRNA isprovided as a pesticide or in a pesticidal formulation to the pestspecies.

Particular suitable target genes are genes that are involved in anessential biological pathway of the target pest species, meaning thatthe target gene is an essential gene to the target pest species and thatgene silencing of the target gene has an adverse effect on the viabilitythe growth, development, feeding, movement, and/or reproduction of thetarget pest species. Suitable target genes are genes associated withinfection, propagation or pathogenesis of the pest species in the host.In another preferred embodiment the sequence of the target RNA in thepest organism comprises sequences of genes which are essential fordevelopment, neural function, reproduction or digestion of the pestorganism.

In accordance with the present invention, any suitable double-strandedRNA fragment capable of directing RNAi or RNA-mediated gene silencing ofa target gene can be used.

As used herein, a “double-stranded ribonucleic acid molecule (dsRNA)”refers to any RNA molecule, fragment or segment containing two strandsforming an RNA duplex, notwithstanding the presence of single strandedoverhangs of unpaired nucleotides.

The double-stranded RNA comprises annealed complementary strands, one ofwhich has a nucleotide sequence which corresponds to a target nucleotidesequence of the target gene to be down-regulated. The other strand ofthe double-stranded RNA is complementary to this target nucleotidesequence.

According to the invention, the “dsRNA” or “double stranded RNA”,whenever said expression relates to RNA that is capable of causinginterference, may be formed from two or more separate polynucleotidestrands which together form a double stranded, folded or assembledstructure which includes at least one double-stranded portion effectivein gene silencing by RNAi. For example, said dsRNA may be formed formtwo separate (sense and antisense) RNA strands that are annealedtogether. In this embodiment, the sense and antisense strands of thedsRNA originate form distinct RNA molecules, wherein at least one of theRNA molecules is a chimeric RNAi molecule as described above, expressedin the same or in another cell, and which RNA molecule may or may notcomprise other domains or sequences which protect it from degradation orwhich direct it to specific locations. The RNA molecules may, whenfolded or assembled, include both double-stranded and single-strandedregions. The dsRNA may also comprise other sequences that are notcomplementary to a target gene or sequence but that have otherfunctions.

Alternatively, the dsRNA may be formed from a single RNA polynucleotidemolecule which includes regions of self-complementarity, such that whenfolded it is capable of forming a structure including one or moredouble-stranded portions (also referred as “dsRNA fragment(s)”)effective in gene silencing by RNAi. For example, the dsRNA may have afoldback stem-loop or hairpin structure wherein the two annealed strandsof the dsRNA are covalently linked. In this embodiment, the sense andantisense strands of the dsRNA are formed from different regions of asingle chimeric RNAi molecule that is partially self-complementary. Thefeatures of “hairpin” or “stem-loop” RNAs for use in RNAi are generallyknown in the art (references: WO 99/53050). The organization of senseand antisense portions making up the double stranded RNA is variable.RNAs having this structure are convenient if the dsRNA is to besynthesized by expression in vivo, for example in a host cell ororganism as discussed below, or by in vitro transcription. Non-limitingexamples of the organization of sequences within a chimeric RNAimolecule are represented in FIG. 55, 59 or 75.

Further, as used herein, a double-stranded ribonucleic acid molecule mayfurther include single stranded RNA molecules forming functionalstem-loop structures, such as small temporal RNAs, short hairpin RNAsand microRNAs, thereby forming the structural equivalent of an RNAduplex with single strand overhangs. The RNA molecule of the presentinvention may be isolated, purified, native or recombinant, and may bemodified by the addition, deletion, substitution and/or alteration ofone or more nucleotides, including non-naturally occurring nucleotides,including those added at 5′ and/or 3′ ends to increase nucleaseresistance.

If the methods of the invention are to be used for controlling growth orinfestation of a pest organism in a host, it is preferred that that thedsRNA is not harmful for organisms other than the target organism(s),and consequently the double-stranded RNA does not share any significanthomology with any host gene, or at least with any essential gene of thehost. In this context, it is preferred that the double-stranded RNAshows less than 30%, more preferably less that 20%, more preferably lessthan 10%, and even more preferably less than 5% nucleic acid sequenceidentity with any gene of the host cell. % sequence identity should becalculated across the full length of the double-stranded RNA sequencecapable of causing RNA interference. If genomic sequence data isavailable for the host organism then it is simple to cross-checksequence identity with the double-stranded RNA using standardbioinformatics tools.

Alternatively, in this context, it is preferred that 21 contiguous basepairs of the dsRNA do not occur in the genome of the host organism.

Preferably, the double-stranded RNA sequence capable of causing RNAinterference does not have 20 contiguous nucleotides in common with asequence of an organism other than the target organism. For example,when the target organism is a plant pathogen, such as a plant parasiticnematode or an insect, the double-stranded RNA does not have 20contiguous nucleotides in common with a nucleotide sequence from a plantor a mammal (a human in particular).

The “target region” of the target pest gene may be any suitable regionof the gene. The target region should comprise at least 17 or 18consecutive nucleotides of the target gene, more preferably at least 19,20 or 21 nucleotide and still more preferably at least 22, 23, 24 or 25nucleotides of the target gene.

The term “complementarity” as used herein relates to DNA-DNA and RNA-RNAcomplementarity as well as to DNA-RNA complementarity. In analogyherewith, the term “RNA equivalent” means that in a DNA sequence(s), thebase “T” may be replaced by the corresponding base “U” normally presentin ribonucleic acids.

The term “nucleotide sequence which is complementary to” means asequence that is complementary to at least part of a nucleotide sequenceof a target gene. The term “complementary” when used in the context ofthe present invention for a dsRNA, means having substantial sequenceidentity to one of the strands of the target gene. In the presentinvention, the complementary sequence will generally comprise anucleotide sequence having more than about 75% sequence identity to thecorresponding sequence of the target gene, however, a higher homologymight produce a more efficient inhibition of expression of the targetgene. Preferably, the sequence identity is about 80%, 85%, 90%, 95%, andeven more preferably more than about 99%. In the context of the presentinvention, the expression “more than about” has the same meaning as “atleast”.

It is most preferred that (at least part of) the double-stranded RNAwill share 100% sequence identity with the target region of the targetpest gene. However, it will be appreciated that 100% sequence identityis not essential for functional RNA inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for RNA inhibition. Theterm “corresponding to”, when used to refer to sequence correspondencebetween the double-stranded RNA and the target region of the targetgene, is therefore to be interpreted accordingly as not absolutelyrequiring 100% sequence identity.

Although the dsRNA must contain a sequence which corresponds to thetarget region of the target gene (i.e. wherein one strand of the dsRNAis complementary to at least part of a target (e.g. pest) nucleotidesequence) it is not absolutely essential for the whole of the dsRNA tocorrespond to the sequence of the target region. For example, the dsRNAmay contain short non-target regions flanking the target-specificsequence, provided that such sequences do not affect performance of thedsRNA in RNA inhibition to a material extent.

In another embodiment, the dsRNA may comprises multiple dsRNA fragments,each fragment comprising annealed complementary strands, one of which iscomplementary to a least part of a target nucleotide sequence to besilenced. Multiple dsRNA fragments as used in the present invention arealso generally referred as to “concatemers”. Thus, the present inventionprovides a chimeric RNAi molecule comprising:

-   -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, and    -   multiple dsRNA fragments (concatemers), each comprising annealed        complementary strands, one of which comprises a nucleotide        sequence which is complementary to at least part of the        nucleotide sequence of a pest target gene.

The term “multiple” in the context of the present invention means atleast two, at least three, at least four, at least five, at least six,etc. and up to at least 10, 15, 20 or at least 30.

Non-limiting examples of suitable concatemers for use in the presentinvention include concatemer cloverleaf, concatemer dumbbell, concatemerhairpin, concatemer stem dsRNA.

In one embodiment, said dsRNA comprises multiple dsRNA fragments thatare complementary to different (e.g. distinct) sequences in one targetgene. In another embodiment, said dsRNA comprises multiple dsRNAfragments that are complementary to different (e.g. distinct) targetgenes. In yet another embodiment, said dsRNA comprises at least onerepeat of one dsRNA fragment. As used herein “one repeat” means twocopies of the same dsRNA fragment. In yet another embodiment, said dsRNAcomprises at least two or three copies, preferably at least four, fiveor six copies, more preferably at least seven, eight, nine ten or morecopies of one (e.g. the same) dsRNA fragment. In other words, saidmultiple dsRNA fragments are repeats of a single dsRNA fragment. In onepreferred embodiment, the dsRNA fragments are not separated bynon-hybridizing RNA fragments. In another embodiment, the dsRNAfragments are separated by a linker or spacer sequence. Preferably, thelinker or spacer sequence is double stranded and the strands arecomplementary, thus also forming a double stranded region. The linkersequence may comprise a short random nucleotide sequence that is notcomplementary to target sequences. In another embodiment, the dsRNAfragments are not separated by a linker, a spacer or a lock sequence asdescribed further.

The present invention encompasses chimeric RNAi molecules comprising atleast one nucleotide sequence recognized by an RNA-binding protein orRNA-binding domain, and one dsRNA fragment comprising annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to a least part of a target nucleotide sequence of a pesttarget gene, and which comprises one or more additional dsRNA fragments,each comprised of annealed complementary strands, wherein at least onecomplementary strands of each dsRNA fragment, comprises eachindependently a nucleotide sequence which is complementary to

-   -   at least part of said nucleotide sequence of said pest target        gene, or    -   at least part of another nucleotide sequence of said pest target        gene, or    -   at least part of the nucleotide sequence of another pest target        gene, or a combination thereof.

It should be clear that the expression “multiple dsRNA” in the chimericRNAi molecule also encompasses chimeric RNAi molecules comprising copiesof one or more dsRNA fragments and further comprising other dsRNAfragments, that are different from the repeated or copied dsRNAfragments. Therefore, the invention also relates to chimeric RNAimolecule comprising in addition to at least one nucleotide sequencerecognized by an RNA-binding protein or RNA-binding domain, one or morerepeats of dsRNA fragments and further comprising at least one dsRNAfragment which is distinct from the repeated fragment(s).

In the concatemer comprising chimeric RNAi molecule, the length of eachof the dsRNA fragments may be at least 17 bp, 18 bp, 19 bp, 20 bp, 21bp, 22 bp, 23 bp, 24 bp, 25 bp or more, for example about 30 bp, about40 bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp,about 100 bp, about 110 bp or about 120 bp. Preferred dsRNA fragments ina concatemer comprising chimeric RNAi molecule have a length between 17and 300 bp, preferably between 21 and 250 bp, preferably between 40 and150 bp, more preferably between 50 and 120 bp or any number in between.

The terms “another target gene” or “a further target gene” are useinterchangeably and mean for instance a second, a third or a fourth,etc. target gene.

According to one preferred embodiment, the dsRNA fragments target atleast one target gene that is essential for viability, growth,development or reproduction of the pest and target at least one geneinvolved in pathogenicity or infectivity. Alternatively, the dsRNAfragments target multiple genes of the same category, for example, thedsRNA fragments target at least two essential genes or at least twogenes involved in pathogenicity or at least two genes involved in any ofthe cellular functions. According to a further embodiment, the dsRNAfragments target at least two target genes, which target genes areinvolved in a different cellular function. For example, the dsRNAfragments target two or more genes involved in protein synthesis (e.g.ribosome subunits), protein degradation (e.g. proteasome subunits),formation of microtubule cytoskeleton (e.g. beta-tubulin gene), and thelike.

The dsRNA fragments in the chimeric RNAi molecule may be combined asfollows:

-   -   a) when multiple dsRNA fragments targeting a single target gene        are combined, they may be combined in the original order (i.e.        the order in which the regions appear in the target gene) in the        chimeric RNAi molecule,    -   b) alternatively, the original order of the fragments may be        ignored so that they are scrambled and combined randomly or        deliberately in any order into the chimeric RNAi molecule,    -   c) alternatively, one single fragment may be repeated several        times, for example from 1 to 10 times, e.g. 1, 2, 3, 4, 5, 6, 7,        8, 9 or 10 times, in the chimeric RNAi molecule, or    -   d) the dsRNA fragments (targeting a single or different target        genes) may be combined in the sense or antisense orientation.

The present invention thus encompasses a chimeric RNAi moleculecomprising at least one nucleotide sequence recognized by an RNA-bindingprotein or RNA-binding domain and multiple dsRNA fragments targetingdifferent target genes which originates from a single target (or pest)species, or wherein said different target genes originates from distincttarget (or pest) species, for instance pest species belonging to thesame (in one embodiment) or to different (in other embodiments) genera,families, orders or even phyla.

The chimeric RNAi molecules comprising such multiple dsRNA fragments andtargeting multiple target genes, are characterized by stacking multipleRNAi capacity, resulting in synergetic effects, and capable oftriggering multiple RNAi effects in the target cell or target organism.

The dsRNA may further contain DNA bases, non natural bases ornon-natural backbone linkages or modifications of the sugar-phosphatebackbone, for example to enhance stability during storage or enhanceresistance to degradation by nucleases.

The double-stranded RNA fragment capable of causing interference willitself preferably be greater than 17 bp in length, preferably 19 bp inlength, more preferably greater than 20 bp, more preferably greater than21 bp, or greater than 22 bp, or greater than 23 bp, or greater than 24bp, or greater than 25 bp in length.

The design and production of suitable chimeric RNAi molecules comprisingdouble stranded RNA for the present invention is documented in theexamples section. Optionally, within the chimeric RNAi molecules of thepresent invention, there may be included one or more moieties capable ofprotecting the double stranded RNA portion (causing RNA interference)against RNA processing. Such moieties and constructs are described in apatent application of applicant having application number 0423659.2 andwhich was filed on Oct. 25, 2004 at the UK Patent Office (and of whichthe publication number will be provided once available) and other patentapplications of applicant having application number U.S. 60/621,800 andwhich was filed on Oct. 25, 2004 and having application number U.S.60/683,551 which was filed on May 5, 2005, at the US Patent andTrademark Office (and of which the publication number will be providedonce available). Both patent applications are incorporated herein intheir entirety. Chimeric RNAi constructs according to the presentinvention may thus comprise different dsRNA core types, optionallycomprising linker types, optionally comprising different lock typesdesigned to protect the dsRNA core against RNA processing in the hostcell expressing the dsRNA construct. In one embodiment of the inventiona sequence capable of protecting the dsRNA against RNA processing isalso referred to as a “lock”. (For the terminology used in thisparticular section, relating to protection of dsRNA, reference is madeto the above-mentioned UK and US patent applications by applicant).

Whenever a stabilized or protected chimeric RNAi molecule is described,the term “core” refers to the dsRNA portion, which core may comprise atleast one dsRNA fragment or which may comprise multiple dsRNA fragments.

The term “dsRNA core” as used herein refers to the core of the dsRNAmolecule. Different dsRNA core types are for example a single stemcomprising one dsRNA, a single stem comprising multiple dsRNA fragments(concatemer) which dsRNA fragments are each independently complementaryto one target gene or to different target sequences of one target geneor are complementary to different target genes.

The term “lock” as used herein refers to a sequence capable ofprotecting the dsRNA or a portion thereof against RNA processing.Different lock types include a GC rich clamp, a short loop of about 4 orof about 5 base pairs, a mismatch lock, or a protein binding RNAstructure such as an IRES, a 5′ region of a virus, an Iron responsiveelement or other RNA motifs that are recognized by proteins.

Other mechanisms to protect the dsRNA against RNA processing may becombined within the chimeric RNAi molecules of the present invention,for example embedding the dsRNA in viroids or in natural unprocessed RNAstructures (such as mi RNA, tRNA, ribosomal RNA, components of thespliceosome or other non-coding RNA's transcribed from RNA polymerase I,II or III promoters). Embedding the dsRNA in a viroid-like dsRNAstructure is described and illustrated for instance in Navarro andFlores (2000 EMBO Journal 19(11) p 2662. The dsRNA may be incorporatedwithin the viroid as such, or in the viroid mutated to avoid internalcleavage (for example by ribozymes) or to avoid translation. Mutationscan be based on information from Dais et al. (1991, NAR 19(8), p 1893).These type of constructs may be transported to the chloroplasts, whereit can receive extra protection against dsRNA processing.

Another mechanism to protect dsRNA from processing is to signal thedsRNA towards an intracellular compartment of the host cell. Forexample, the dsRNA can be compartmentalized in an intermediate hostcell, before it is transferred to the target host cell. In particular,the chimeric RNAi molecule may be compartmentalized in a plant cell, forexample, it may be located in the chloroplast, mitochondrion or plastid,before it is transferred to the plant pest species, for example theplant pest nematode or insect. Compartmentalization may occur in avariety of ways, such as for example via the use of viroid structures,or via the use of signal sequences, for example chloroplast,mitochondrial or plastid signal sequences. These organelles are fromprokaryotic origin and may offer a protective environment away from theplant RNA processing machinery.

The term “linker” as used herein for the chimeric RNAi molecule refersto a molecule enabling linking of a lock to a dsRNA core. Differentlinker types are conditionally self-cleaving RNA moieties such aslinkers that are cleaved at low pH or at high pH or that are cleaved inhydrophobic conditions, or are one of an intron, or a non-complementaryRNA sequence. Optionally, the chimeric RNAi molecule may comprise aninterstem base pairing moiety or can be in the form of a triple RNA. Ina preferred embodiment, the multiple dsRNA fragments of the chimericRNAi molecule are connected without linker. In another embodiment, alinker is present between the at least one nucleotide sequencerecognized by an RNA-binding protein or RNA binding domain and the dsRNAin the chimeric RNAi molecule. In still another embodiment, the multipledsRNA fragments of the chimeric RNAi molecule are connected by one ormore linkers.

In a particular embodiment, the linkers may be used to disconnectsmaller dsRNA regions in the pest organism. Advantageously, in thissituation the linker sequence may promote division of a long dsRNA intosmaller dsRNA under particular circumstances, resulting in the releaseof separate dsRNA fragments under these circumstances and leading tomore efficient gene silencing by these smaller dsRNA fragments.Non-limiting examples of suitable conditionally self-cleaving linkersare RNA sequences that are self-cleaving at suitable pH conditions.Suitable examples of such RNA sequences are described by Borda et al.(Nucleic Acids Res. 2003 May 15, 31(10):2595-600), which document isincorporated herein by reference. This sequence originates form thecatalytic core of the hammerhead ribyozome HH16. Alternatively, thelinkers are self cleaving in the endosomes. This may be advantageouswhen the chimeric RNAi molecule of the invention is taken up by the pestvia endocytosis or transcytosis, and are therefore compartmentalized inthe endosomes of the pest. The endosomes may have a low pH environment,leading to the cleavage of the linker. Linkers that are self-cleaving inhydrophobic conditions are particularly useful in chimeric RNAimolecules of the present invention when used to be transferred from onecell to another via the transit in a cell wall, for example whencrossing the cell wall of a pest. Particular plant pest organisms ofinterest for application of this technique are plant parasitic fungi orplant parasitic viruses or bacteria.

An intro may be used as a linker. An “intron” as used herein may be anynon-coding RNA sequence of a messenger RNA. Particular suitable intronsequences for the chimeric RNAi molecules of the present invention are(1) U-rich (35-45%); (2) have an average length of 100 bp (varyingbetween about 50 and about 500 bp) which base pairs may be randomlychosen or may be based on known intron sequences; (3) strat at the 5′end with -AT:GT- or -CG:GT- and/or (4) have at their 3′ end -AG/GC- orAG:AA.

According to the invention, a linker sequence may be present between thedsRNA fragments or not. Preferably, no linker sequence is presentbetween the dsRNA fragments. For instance when the dsRNA comprising thedsRNA fragments is chemically synthesized, the dsRNA fragments may bedirectly adjacent to each other, without the presence of non-targetsequences. When present, the linker may for instance comprise a shortrandom nucleotide sequence that is not complementary to target sequencesbut that is the result of the cloning.

A by itself non-complementary RNA sequence, ranging from about 1 basepair to about 10000 base pairs, for instance of at least 10, 20, 30, 50,60, 70, 80, 90, 100, 200, 500, 1000, 1500, 2000, 3000, 10000 base pairs,or any number in-between, may also be used as a linker.

Choice of Target Gene(s) to be Targeted by a Chimeric RNAi Molecule or aComplex Thereof Comprising an RNA Delivery Molecule According to theInvention

The choice of target gene(s) to be targeted by a chimeric RNA moleculedepends on the choice of target gene which is to be silenced in thetarget organism or organisms in order to achieve the desired effect ofpest control. For the chimeric RNA molecule designed herein below thetarget gene(s) was (were) chosen from one or more of the followingcategories of genes:

-   -   1. “essential” genes encompass genes that are vital for one or        more target organisms and result in a lethal or severe (e.g.        movement, feeding, paralysis, drinking, fertility) phenotype        when silenced.    -   2. “pathogenicity genes” are genes that are involved in the        pathogenicity or infectivity of the pest.    -   3. “pest specific” genes encompass genes that have no        substantial homologous counterpart in non-pest organisms as can        be determined by bioinformatics homology searches, for example        by BLAST searches. The choice of a pest specific target gene        results in a species-specific RNAi effect, with no effect or no        substantial (adverse) effect in non-target organisms.    -   4. “conserved pathway” genes encompass genes that are involved        in the same biological pathway or cellular process, or encompass        genes that have the same functionality in different species        resulting in a specific and potent RNAi effect and more        efficient pest control.    -   5. According to the present invention, the chimeric RNA        molecules target genes inducing improved        delivery/uptake/endocytosis in the pest, such as for example        chitin synthase genes, genes encoding peritrophic membrane        proteins, excreted RNAses, proteins involved in the secretion of        Rnase in the gut; tight junction genes, septate junction genes,        genes encoding proteins involved in the acidification of the gut        (especially for lepidoteren insect, such as ion channels) and        any proteins involved in the maintenance and/or regeneration of        gut epithelium.

Combination of Multiple Target Sequence or Multiple Target Species

The RNA delivery molecules, chimeric RNAi molecules, complexes thereofand methods of the present invention are particularly useful to targetmultiple sequences simultaneously. These multiple sequences mayoriginate from one target gene. Alternatively, the multiple targetsequences may originate from multiple target genes. These multipletarget genes may originate from one and the same pest species.Alternatively, these multiple target genes may originate from differentpest species from the same or different order. Therefore, one chimericRNAi molecule of the present invention, for example in the form of aconcatemer cloverleaf, a concatemer stem, or a concatemer hairpin, maysimultaneously target multiple sequences originating from the same andfor multiple target genes of the same and or different pest species,such as from nematodes, insects, bacteria and/or fungi.

According to one particular embodiment of the present invention, the RNAdelivery molecules, chimeric RNAi molecules, or complexes thereoftargets multiple target genes originating from multiple species. Forexample, a chimeric RNAi molecule may target multiple genes frommultiple plant pest organisms, and by expressing the chimeric RNAimolecule in the plant, the plant acquires resistance against multipleplant pests simultaneously. Similarly, a plant or a surface or substancesusceptible to pest infestation may be sprayed with a composition (orthe like) comprising the RNA delivery molecules, chimeric RNAimolecules, or complexes thereof, thereby protecting the plant or thesurface or substance against infestation from multiple pests. Forexample, the plant acquires resistance against nematodes and insects, oragainst nematodes, insects and/or fungi. Also the chimeric RNAi moleculeallows the plant to acquire resistance against multiple nematodes of adifferent genus, family, order or class, and/or against insects of adifferent genus, family or order, and/or against fungi of a differentgenus, family or order.

In another particular embodiment of the present invention, the chimericRNAi molecule targets multiple target genes originating from differentspecies from the same order. For example, one chimeric RNAi moleculewhich targets genes of different bacterial, viral, fungal, insect ornematode species, may be used as an effective and broad spectrumbacteria, virus, fungus, insect killer or broad spectrum nematodekiller. Combination of dsRNA fragments with at least one nucleotidesequence recognized by an RNA-binding protein or RNA-binding domain intoone chimeric RNAi molecule according to the present invention, whereinsaid dsRNA fragments are targeting multiple target sequences fromdifferent pest species is favorable to enlarge the pest species spectrumof the RNAi effect of the dsRNA molecules.

In another particular embodiment of the present invention, the chimericRNAi molecule targets multiple target genes originating from the sameorganism, for example from the same pest species. Such a constructoffers the advantage that several weak target genes from the sameorganism can be silenced together to efficiently control the pestorganism, while silencing one or more of the weak target genesseparately is not effective to control the pest. Also, several strongtarget genes from the same organism can be silenced simultaneously, inorder to further improve the efficacy of the pest control, or to avoidthe occurrence of resistance of the pest organisms by mutation.

C. Target and Pest Organisms

The terms “target organism” or “target species” or “pest organism” or“pest species” are used herein as synonym and refer to any organism orspecies which needs to be killed or paralyzed. Suitable target speciesare chosen from the group comprising fungi, insects, mites, protozoa,metazoa (comprising nematodes), algae, plants, animals (includingmammals). Most suitable for the methods of the present invention aretarget species which are pest organisms. Preferably, in the context ofthe present invention, the term “target species” or “pest species”refers to plant pest organisms such as nematodes, insects and fungi andpreferably refers to any organism classified in the taxonomical group ofthe Insecta or of the Nematoda, or of the Fungi.

Nematodes

“Nematodes” as used herein comprises species of the order Nematoda. Manyspecies of nematodes are parasitic and cause health problems to humansand animals (for example species of the orders Ascaradida, Oxyurida,Strongylida, Stronglyloides and Trichocephalida), as well as to plantsand fungi (for example species of the orders Aphelenchida, Tylenchida adothers). Preferably, “nematodes” as used herein refers to plantparasitic nematodes and nematodes living in the soil. Plant parasiticnematodes include, but are not limited to, ectoparasites such asXiphinema spp., Longidorus spp., and Trichodorus spp.; semiparasitessuch as Tylenchulus spp.; migratory endoparasites such as Pratylenchusspp., Radopholus spp., and Scutellonerna. spp.; sedentary parasites suchas Heterodera spp., Globodera spp., and Meloidogyne spp., and stem andleaf endoparasites such as Ditylenchus spp., Aphelenchoides spp., andHirshmaniella spp. According to a preferred embodiment of the invention,the nematodes are plant parasitic nematodes, in particular rootparasitic soil nematodes such as, for example, those of the generaHeterodera and Globodera (cyst-forming nematodes) and Meloidogyne (rootknot nematodes). The RNA constructs of the present invention areparticularly suitable to control harmful species of the genusMeloidogyne, such as for example, Meloidogyne incognita, and of thegenus Heterodera, such as for example, Heterodera glycines (soybean cystnematode) and also of the genus Globodera, such, as for example,Globodera rostochiensis (potato cyst nematode) and also representativesof migrating endoparasites, such as, for example, Pratylenchus penetransor Radopholus similes and representatives of ectoparasites, such as, forexample, Trichodorus spp. and Xiphinema spp. However, the use of the RNAconstructs according to the invention is in no way restricted to thesegenera, but also extends in the same manner to other nematodes.

Fungi

“Fungi” as used herein comprises all species of the order Fungi.According to a preferred embodiment of the invention, the target geneoriginates from a plant parasitic fungus such as Magnaporthe oryzae(rice blast, formerly Magnaporthe grisae; anamorph Pyricularia oryzaeCav. and Pyricularia grisae); Rhizoctonia spp., particularly Rhizoctoniasolani and Rhizoctonia oryzae; Gibberella fujikuroi; Sclerotinium spp.;Helminthosporium sigmoideum; Pythium spp.; Alternaria spp., particularlyAlternaria solani; Fusarium spp., particularly Fusarium solani andFusarium germinearum; Acremoniella spp.; Leptosphaeria salvinii;Puccinia spp., particularly Puccinia recondita and Puccinia striiformis;Septoria nodorum; Pyrenophora teres; Rhincosporium secalis; Erysiphespp., particularly Erysiphe graminis; Cladosporium spp.; Pyrenophoraspp.; Tilletia spp.; Phytophthora spp., particularly Phytophthorainfestans; Plasmopara viticola; Uncinula necator, Botrytis cinerea;Guiguardia bidwellii; C. viticola; Venturia inaequafis; Erwiniaarmylovora; Podosphaera leucotricha; Venturia pirina; Phakospora sp(soybean rust), Ustilago maydis (corn smut).

Insects

“Insects” as used herein comprises all insect species. In an embodimentsaid insect species comprise species of the order Lepidoptera. Accordingto a preferred embodiment of the invention, the insects are insects thatdamage plants, comprising amongst others Leptidopteran insect pests,such as Heliothis spp., Helicoverpa spp., Spodoptera spp., Ostriniaspp., Pectinophora spp, Agrotis spp., Scirphophaga spp., Cnaphalocrocisspp., Sesamia spp, Chilo spp., Anticarsia spp., Pseudoplusia spp.,Epinotia spp., and Rachiplusia spp., preferably Heliothis virescens,Helicoverpa zea, Helicoverpa armigera, Helicoverpa punctera, Ostrinianubilafis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophoragossypiella, Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamiainferens, Chilo partellus, Anticarsia gemmatalis, Pseudoplusiaincludens, Epinotia aporema and Rachiplusia nu. e.g. Examples ofpreferred Insecta include, but are not limited to, members of the ordersColeoptera (Anobium, Ceutorhynchus, Rhynchophorus, Cospopolites,Lissorhopterus spp., Lissorhopterus oryzophilus, Meligethes,Echinocnemus squamos, Hypothenemus, Hylesinus, Acalymma, Lema,Psylliodes, Leptinotarsa, Gonocephalum, Agriotes, Dermolepida,Heteronychus, Phaedon, Tribolium, Sitophilus spp., Sitophilus zeamais,Diabrotica spp. (Diabrotica virgifera virgifera, Daibroticaundecimpunctata howardi, Diabrotica barbell), Oulema oryzae, Chaetocnemapulicaria, Epilachna varivestis, Cerotoma trifurcata, Leptinotarsadecemlineata, Anthonomus spp., Anthonomus grandis, or Anthrenus spp.),Lepidoptera (e.g. Ephestia, Mamestra, Earias, Pectinophora, Ostrinia,Trichoplusia, Pieris, Laphygma, Agrotis, Amathes, Wiseana, Tryporyza,Diatraea, Sporganothis, Cydia, Archips, Plutella, Chilo, Heliothis,Helicoverpa (especially Helicoverpa armigera), Spodoptera or Tineolassp.), Diptera (e.g. Musca, Aedes, Anopheles, Culex, Glossina, Simulium,Stomoxys, Haematobia, Tabanus, Hydrotaea, Lucilia, Chrysomia,Callitroga, Dermatobia, Gasterophilus, Hypoderma, Hylemyia, Atherigona,Chlorops, Phytomyza, Ceratitis, Liriomyza, and Melophagus spp.),Phthiraptera, Hemiptera (e.g. Laodelphax striatellus, Sogatellafurcifera, Rhopalosiphum maidis, Macrosiphum euphorbiae, Aphis spp.(Aphis gossypii, Aphis glycines), Bemisia spp., Bemisia tabaci,Phorodon, Aeneoplamia, Empoasca spp. (Empoasca fabae, Empoasca solana),Parkinsiella, Pyrilla, Aonidiella, Coccus, Pseudococcus, Helopeltis,Lygus, Dysdercus, Oxycarenus, Nezara, Aleurodes, Triatoma, Rhodnius,Psylla, Myzus spp., Myzus persicae, Megoura, Phylloxera, Adelyes,Nilaparvata spp., Nilaparvata lugens, Nephrotettix spp., Nephotettixvirescens, or Cimex spp.), Orthoptera (e.g. Locusta, Gtyllus,Schistocerca or Acheta spp.), Dictyoptera (e.g. Blattella, Periplanetaor Blatta spp.), Hymenoptera (e.g. Athalia, Cephus, Atta, Lasius,Solenopsis or Monomorium spp.), Isoptera (e.g. Odontotermes andReticulitermes spp.), Siphonaptera (e.g. Ctenocephalides or Pulex spp.),Thysanura (e.g. Lepisma spp.), Dermaptera (e.g. Forficula spp.) andPsocoptera (e.g. Peripsocus spp.) and Thysanoptera (e.g. Thrips tabaci).

Bacteria

“Bacteria” that damage plants and that can be controlled with theconstructs and methods of the present invention are for exampleAgrobacterium ssp.; Arachnia ssp.; Clavibacter ssp.; Corynebacteriumssp.; Erwinia ssp.; Fusobacterium ssp.; Hafnia ssp.; Pseudomonas ssp.;Spiroplasma ssp.; Streptomyces ssp.; Xanthomonas ssp.; Xylella ssp. andXylophilus ssp.

Viruses

“Viruses” that damage plants and that can be controlled with theconstructs and methods of the present invention are for example Africancassava mosaic virus; Alfalfa mosaic virus; American plum line patternvirus; Andean potato latent virus; Andean potato mottle virus; Applechlorotic leaf spot virus; Apple mosaic virus; Apple stem groovingvirus; Arabis mosaic virus; Arracacha virus B, oca strain; Asparagusvirus 2; Australian grapevine viroid; Avocado sunblotch viroid; Barleymild mosaic virus; Barley stripe mosaic virus; Barley yellow dwarfvirus; Barley yellow mosaic virus; Bean common mosaic virus; Bean goldenmosaic virus; Bean leaf roll virus; Bean pod mottle; Bean yellow mosaicvirus; Bearded iris mosaic virus; Beet curly top virus; Beet leaf curlvirus; Beet mosaic virus; Beet necrotic yellow vein virus; Beet pseudoyellows virus; Beet western yellows virus; Beet yellow stunt virus;Belladona mottle virus; Black rasberry latent virus; Blight (etanalogues/en analoge); Blueberry leaf mottle virus; Broad bean wiltvirus; Bromoviruses; Cacao swollen shoot virus; Cacao yellow mosaicvirus; Cactus virus X; Cadan-cadang viroid; Camation cryptic virus;Camation etched ring virus; Camation latent virus; Camation mottlevirus; Camation necrotic fleck virus; Camation ringspot virus; Camationvein mottle virus; Cassava common mosaic virus; Cauliflower mosaicvirus; Chemy leafroll virus; Chemy rasp leaf virus; Chemy rasp leafvirus (American); Chemy rugose virus; Chrysanthemum B virus;Chrysanthenum stunt viroid; Citrus exocortis viroid; Citrus leaf rugosevirus; Citrus mosoie virus; Citrus tristeza virus (European isolates);Citrus tristeza virus (non-European isolates); Citrus variegation virus;Citrus veinenation woody gall; Citrus viroids; Clover Yellow vein virus;Cocksfoot mild mosaic virus group; Cocksfoot streak virus; Cowpea mildmottle virus; Cucumber mosaic virus; Cucumber yellows virus; Cucumovirussatellites; Cymbidium mosaic virus; Dahlia mosaic virus; Dasheen mosaicvirus; Dianthoviruses; Echtes Ackerbohnenmosaic virus; Elderberrycarlavirus; Euphorbia mosaic virus; Florida tomato virus; Grapevinealgerian latent virus; Grapevine bulgarian latent virus; Grapevinefanleaf virus; Grapevine flavescence dorée mycoplasm; Grapevine leafrollassociated virus (I to V); Grapevine tunusian ringspot virus; Grapevinevirus A; Grapevine yellow speckle viroids (I & II); Grapewine chromemosaic virus; Heracleum latent virus; Hippeastrum mosaic virus;Honeysuckle latent virus; Hop (American) latent virus; Hop latent virus;Hop mosaic virus; Hop stunt viroids; Hop virus A; Hop virus C; Hydrangearingspot virus; Iliaviruses; Iris mild mosaic virus; Leek yellow stripevirus; Leprosis; Lettuce infectious yellows virus; Lettuce mosaic virus;Lilac chlorotic leafspot virus; Lilac ring mottle virus; Lillysymptomless virus; Luteovirus satellites; Maize dwarf mosaic virus;Maize streak virus; Marafiviruses; Melon necrotic spot virus; Myrobolanlatent ringspot virus; Narcissus latent virus; Narcissus mosaic virus;Narcissus tip necrosis virus; Narcissus yellow stripe virus; Oat goldenstripe virus; Oat mosaic virus; Odontoglossum ringspot virus; Olivelatent ringspot virus; Onion yellow dwarf virus; Papaya mosaic virus;Papaya ringspot virus; Parsnip yellow fleck virus; Pea early browningvirus; Pea enation mosaic virus; Pea seed borne mosaic virus; Peachmosaic virus (American); Pear decline mycoplasm; Pelargonium leaf curlvirus; Pepper mild tigré virus; Plant reoviruses; Plum line patternvirus (American); Plum pox virus; Poinsettia mosaic virus; Poplar mosaicvirus; Potato aucuba mosaic virus; Potato black ringspot virus; Potatoleafroll virus; Potato leafroll virus (non European isolates); Potatomop-top virus; Potato spindle tuber viroid; Potato virus A; Potato virusA (non European isolates); Potato virus M; Potato virus M (non europeanisolates); Potato virus S; Potato virus S (non European isolates);Potato virus T; Potato virus X; Potato virus X (non European isolates);Potato virus Y; Potato virus Y (non European isolates); Potato yellowdwarf virus; Potato yellow mosaic virus; Prune dwarf virus; Prunusnecrotic ringspot virus; Raspberry bushy dwarf virus; Raspberry leafcurl virus (American); Raspberry ringspot virus; Raspberry veinchlorosis virus; Red clover mottle virus; Red clover vein mosaic virus;Ribgrass mosaic virus; Rice stripe virus group; Rubus yellow net virus;Saguro cacao virus; Satellites (andere dan geciteerde); Satsuma dwarfvirus; Shallot latent virus; Sharka virus; Sobemoviruses; Sowbane mosaicvirus; Sowthistle yellow vein virus; Spinach latent virus; Squash leafcurl virus; Stolbur mycoplasm; Strawberry crinkle virus; Strawberrylatent C virus; Strawberry latent ringspot virus; Strawberry mild yellowedge virus; Strawberry vein banding virus; Sugar beet yellows virus;Tater leaf virus; Tobacco etch virus; Tobacco mosaic virus; Tobacconecrosis virus; Tobacco rattle virus; Tobacco ringspot virus; Tobaccostreak virus; Tobacco stunt virus; Tomato apical stunt viroid; Tomatoaspermy virus; Tomato black ring virus; Tomato bunchy top viroid; Tomatobushy stunt virus; Tomato mosaic virus; Tomato planta macho viroid;Tomato ringspot virus; Tomato spotted wilt virus; Tomato yellow leafcurt virus; Tulare apple mosaic virus; Tulip breaking virus; Turnipcrinkle virus satellites; Turnip crinkle virus; Turnip mosaic virus;Turnip yellow mosaic virus; Tymoviruses; Velvet tobacco mottle virus;other Viroids; Watermelon mosaic virus 2; Wheat dwarf virus; Wheatsoil-bome mosaic virus; Wheat spindle steak mosaic virus; Wheat yellowmosaic virus; White clover mosaic virus; Yam mosaic virus; Zucchiniyellow fleck virus; and Zucchini yellow mosaic virus.

The pest organism can be any species. Preferably, the pest organism isany insect or nematode of economic importance, such as, for example,organisms that cause disease, a household pest, or an agricultural pest,or that are associated with plants diseases, e.g. in corn, potatoes,soybeans, sugarbeets, turf, trees, orchards and vineyards, gardenvegetables, etc.

The pest organism can be at any stage of development, however, it ispreferred that when the organism is an insect, it is in a larval oradult developmental stage when the dsRNA is delivered.

D. Nucleotide Sequences, Vectors and Host Cells

In another embodiment, the invention relates to nucleic acids encodingthe RNA delivery molecule and encoding the chimeric RNAi molecules asdescribed herein.

Vectors

According to a further aspect of the present invention, there areprovided expression constructs, also referred herein as recombinant DNAconstructs, to facilitate introduction into a host cell and for instancea plant cell and/or facilitate expression and/or facilitate maintenanceof the nucleotide sequence encoding the RNA delivery molecules and/orthe chimeric RNAi molecules according to the invention. The expressionconstructs may be inserted into a plasmid or a vector, which may becommercially available.

According to an embodiment of the present invention, the expressionconstruct is an expression vector, suitable for transformation into hostorganisms such as yeast, bacteria, fungi or plants or plant cells andsuitable for maintenance and expression of an RNA delivery molecule or achimeric RNAi molecule according to the present invention in atransformed host cell. An “expression vector” is a construct that can beused to transform a selected host cell and provides for expression of acoding sequence in the selected host. Expression vectors can forinstance be cloning vectors, binary vectors or integrating vectors. Theinvention thus also relate to a vector comprising any of the nucleicacids described above. Said vector may further comprise regulatorysequences for controlling expression of the nucleic acid in said hostcell.

According to one embodiment of the present invention, the expressionconstruct is a plant expression vector, suitable for transformation intoplants and suitable for maintenance and expression of an RNA deliverymolecule or a chimeric RNAi molecule according to the present inventionin a transformed plant cell. The invention thus also relates to a vectorcomprising any of the nucleic acids described above. Said vector mayfurther comprise regulatory sequences for controlling expression of thenucleic acid in a plant cell.

The terms “regulatory sequences” and “control sequence” used herein areto be taken in a broad context and refer to regulatory nucleic acidsequences capable of driving and/or regulating expression of thesequences to which they are ligated and/or operably linked. The controlsequences differ depending upon the intended host organism and upon thenature of the sequence to be expressed. For expression of a protein inprokaryotes, the control sequences generally include a promoter, aribosomal binding site, and a terminator. In eukaryotes, controlsequences generally include promoters, terminators and, in someinstances, enhancers, and/or 5′ and 3′ untranslated sequences. The term‘control sequence’ is intended to include, at a minimum, all componentsnecessary for expression, and may also include additional advantageouscomponents. According to one embodiment of the present invention, thecontrol sequence is operable in a plant; preferably the control sequenceis a sequence derived from a plant sequence. The term “control sequence”encompasses a promoter or a sequence capable of activating or enhancingexpression of a nucleic acid molecule in a cell, tissue or organ.

Promoters useful for the expression of dsRNA are a promoter from an RNAPoll, an RNA PollI, an RNA PollII, T7 RNA polymerase or SP6 RNApolymerase. These promoters are typically used for in vitro-productionof dsRNA, which dsRNA is then included in an antipesticidal agent, forexample in an anti-pesticidal liquid, spray or powder.

Examples of promoters suitable for the constructs and methods accordingto the present invention are constitutive plant promoters, such as theCaMV35S promoter, doubled CaMV35S promoter, GOS2 promoter, Figwortmosaic viruse (FMV) 34S promoter, rubisco promoter, actin promoter orubiquitin promoter.

In order to improve the transfer of the dsRNA from the plant cell to theplant pest, the plants preferably express the dsRNA in plant partseasily accessible to the plant pest. Preferred tissues to express thedsRNA in are the roots, leafs, stems, rhizomes, shoots, tubers, anthers,petioles, seeds, flowers, fruits. Therefore, tissue-preferred promotersmay be used, such as a root specific promoter or a leaf specificpromoter. Suitable examples of a root preferred promoter are PsMTA(Fordam-Skelton, A. P., et al., 1997 Plant Molecular Biology 34:659-668), Class III Chitinase promoter, etc. . . . Examples of leaf- andstem-specific or photosynthetic tissue-specific promoters that are alsophotoactivated are promoters of two chlorophyll binding proteins (cab1and cab2) from sugar beet (Stahl D. J., et al., 2004 BMC Biotechnology2004 4:31), ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS(Nomura M. et al., 2000 Plant Mol. Biol. 44: 99-106), A (gapA) and B(gapB) subunits of chloroplast glyceraldehyde-3-phosphate dehydrogenase(Conley T. R. et al. 1994 Mol. Cell. Biol. 19: 2525-33; Kwon H. B. etal. 1994 Plant Physiol. 105: 357-67), promoter of the Solanum tuberosumgene encoding the leaf and stem specific (ST-LS1) protein (Zaidi M. A.et al., 2005 Transgenic Res. 14:289-98), stem-regulated,defense-inducible genes, such as JAS promoters (patent publication no.20050034192/US-A1), flower-specific promoters such as chalcone synthasepromoter (Faktor O. et al. 1996 Plant Mol. Biol. 32: 849) andfruit-specific promoters such as that of RJ39 from strawberry (WO 9831812). Other suitable promoters are pathogen-induced promoters, such asnematode induced plant promoters, or feeding-site specific promoters,examples of which are Wun-1 (Hansen et al. 1996. Physiol. Mol. Plant.Pathol. 48: 161-170); Lea-14, Lemmi 9 (Van der Eycken W et al. Plant J.1996 9(1): 45-54; Escobar C et al. Mol Plant Microbe Interact. 1999,12(5):440-9), pin-2 (Keil et al. 1989. EMBO J. 8:1323-1330) and TobRB7(Opperman et al. 1994. Science, 263: 221-223). According to oneembodiment of the invention, the vectors comprise a constitutivepromoter. According to another embodiment of the invention, the vectorscomprise an inducible promoter. According to another embodiment of theinvention, the vectors comprise a tissue-specific promoter, for instancea root-specific promoter in case of alleviating pest infestations wherethe pest predominantly feeds on the roots of the plant, or for instancea leaf-specific promoter in case of alleviating pest infestations wherethe pest predominantly feeds on the leafs of the plant. Promoters whichinitiate transcription only in certain tissues or cells are hereinreferred to ‘tissue-specific or “cell-specific” promoters, respectively.In addition, the present invention relates to a vector according to theinvention wherein the promoter is selected from the group comprisingtissue specific promoters such as any selected from the group comprisingroot specific promoters of genes encoding PsMTA Class III Chitinase,photosynthetic tissue-specific promoters such as promoters of cab1 andcab2, rbcS, gapA, gapB and ST-LS1 proteins, JAS promoters, chalconesynthase promoter and the promoter of RJ39 from strawberry.

Optionally, one or more transcription termination sequences may also beincorporated in the expression construct. The term “transcriptiontermination sequence” encompasses a control sequence at the end of atranscriptional unit, which signals 3′ processing and poly-adenylationof a primary transcript and termination of transcription. Additionalregulatory elements, such as transcriptional or translational enhancers,may be incorporated in the expression construct.

The expression constructs of the invention may further include an originof replication which is required for maintenance and/or replication in aspecific cell type. One example is when an expression construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to the f1-ori and colE1 oh.

The expression construct may optionally comprise a selectable markergene. As used herein, the term “selectable marker gene” includes anygene, which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with an expression construct of theinvention.

Suitable markers may be selected from markers that confer antibiotic orherbicide resistance or visual markers. Examples of selectable markergenes include genes encoding neomycin phosphotransferase (nptII),hygromycin phosphotransferase (hpt) or Basta. Further examples ofsuitable selectable marker genes include resistance genes againstampicillin (Ampr), tetracydine (Tcr), kanamycin (Kanr),phosphinothricin, and chloramphenicol (CAT). Other suitable marker genesprovide a metabolic trait, for example manA. Visual marker genes mayalso be used and include for example beta-glucuronidase (GUS),luciferase and Green Fluorescent Protein (GFP).

Expression of Chimeric RNAi Molecules in Plant Organelles

According to a preferred embodiment of the invention, the RNA deliverymolecule and/or the chimeric RNAi molecules are expressed in plant cellorganelles to protect dsRNA from processing.

According to a further embodiment, the nucleic acids encoding the RNAdelivery molecule and/or the chimeric RNAi molecules may furthercomprise an in frame signal sequence for directing the encodedpolypeptide to a plant organel, such as a chloroplast, a plastide, or amitochondrion, for instance signaling the chimeric RNAi molecule towardsan intracellular compartment of the host cell in another mechanism toprotect dsRNA from processing

For example, the RNA delivery molecule, and/or the chimeric RNAimolecule, and/or the complex between the RNA delivery molecule and thechimeric RNAi molecule can be compartmentalized in an intermediate hostcell or organism before it is transferred to the target host cell, e.g.a pest cell. It shall be understood that the RNA delivery molecule andthe chimeric RNAi molecule may be directed to the same or to differentorganelles of an intermediate host cell.

In particular, the RNA delivery molecule, and/or the chimeric RNAimolecule, and/or the complex between the RNA delivery molecule may becompartmentalized in a plant cell, for instance it may be located in thechloroplast, mitochondrion or plastid, before it is transferred to theplant pest species, for example a plant pest nematode or insect.Compartmentalization may occur in a variety of ways, such as for examplevia the use of viroid structures, or via the use of signal sequences asdescribed above, for example chloroplast, mitochondrial or plastidsignal sequences. These organelles are form prokaryotic origin and mayoffer a protective environment away from the plant RNA processingmachinery.

A major advantage of compartmentalization of the RNA delivery molecule,and/or the chimeric RNAi molecule, and/or the complex between the RNAdelivery molecule thus include that the molecules of the complex areprotected from nuclear/cytoplasmic processing (dicing) of dsRNA. Inaddition, compartmentalization provides for an accumulation of dsRNAsequences.

Compartmentalized Expression of Sense and Antisense Target RNA

In yet another mechanism to protect the dsRNA contained within thechimeric RNAi molecule from RNA processing is to express sense andantisense separately and to target them to different locations withinthe host cell or organism that expresses the sense and the antisensestrands. In the context of the present invention, the sense or theantisense strand is part of the chimeric RNAi molecule. In thisembodiment, sense and antisense RNA fragments corresponding to aselected gene of a particular pest species are cloned behind differentpromoters driving expression (i) separate plant tissues or (ii) withinthe same cell but in separate cellular compartments. These promoters aretissue or organel specific and allow strong simultaneous expression indifferent cellular compartments or in adjacent tissues.

For example, the sense and antisense strands may be targeted todifferent plant tissue or cell types. For example, in a leaf the sensestrand may be expressed in the nerve cells while the antisense isexpressed in the palisade tissue. This may be achieved by usingdifferent promoters to drive the expression of the sense and theantisense strands. The advantage of this technique is that the sense andantisense strands never come together in the plant cell, and thereforeno degradation or auto-silencing or RNA interference can occur withinthe plant by Dicer. When the pest organism feeds on the plant, thestrands are set free and mixed, allowing the annealing of the dsRNA inthe gut lumen and then base pairing between the sense and antisensestrands may occur to form long dsRNA. Subsequently, this dsRNA may betaken up efficiently and leads to the desired RNAi response, leading todegradation of the target mRNA in the pest and death of the pest. Thisapproach can be accomplished by feeding the pest with two bacterialstrains for instance provided in a composition, one strain producing thesense, the other producing the antisense strand.

Host Cells

In another embodiment, the present invention relates to a host cell ororganism comprising a nucleic acid or a vector as defined herein.Examples of host cells which may be used in accordance with the presentinvention include a bacterial, yeast, fungal, or plant cell. Host cellsmay be prokaryotic cells such as E. coli and A. tumefaciens, or may beeukaryotic cells such as yeast, or plant cells. It is preferred thathost cells are monocotyledonous or dicotyledonous plant cells.

Accordingly, the present invention also encompasses a cell, e.g. a hostcell, comprising any of the chimeric RNAi molecules, RNA deliverymolecule, dsRNA, nucleic acid or a vector as defined herein. Theinvention further encompasses prokaryotic cells (such as, but notlimited to, gram-positive and gram-negative bacterial cells) andeukaryotic cells (such as, but not limited to, yeast cells or plantcells). Preferably, said cell is a bacterial cell or a plant cell. Thepresent invention also encompasses a transgenic plant, reproductive orpropagation material for a transgenic plant comprising such a plantcell.

The vector or nucleic acid molecule according to the invention mayeither be integrated into the genome of the host cell or it may bemaintained in some form extrachromosomally.

E. Complex Between RNA Delivery Molecule and the Chimeric RNAi Molecule

In another aspect, the present invention relates to a complexcomprising:

-   -   an RNA delivery molecule as defined herein, and    -   at least one chimeric RNAi molecule comprising        -   at least a double-stranded RNA comprising annealed            complementary strands, one of which has a nucleotide            sequence which is complementary to at least part of a target            nucleotide of a target gene of said pest species (i.e.            capable of causing RNAi interference) and        -   a sequence recognized by an RNA binding protein or RNA            binding domain, for instance as represented in any of SEQ ID            NOs 15 to 40, or as represented in FIG. 41 or 42.

In another embodiment, the present invention relates to a complexcomprising:

-   -   an RNA delivery molecule as defined herein, and    -   at least one chimeric RNAi molecule as represented by any of SEQ        ID NOs 41 to 52 or any of SEQ ID NOs 54 to 56, or any of SEQ ID        NOs 57 to 71, or any of SEQ ID NOs 72 to 86,        provided that the RNA delivery molecule is recognized and may        bind to the RNA binding protein or RNA binding domain of the RNA        delivery molecule.

It is to be understood that said complex is provided in such a conditionthat binding may occur between said RNA delivery molecule and saidchimeric RNAi molecule. This complex is also referred to in the presentinvention as the “protein-RNA complex” or the “complex between the RNAdelivery molecule and the chimeric RNAi”, or the “RNA vehicle complex(RVC)”.

It should be understood that in the above-described complexes of theinvention comprising both an RNA delivery molecule and a chimeric RNAimolecule, the sequences of the molecules should be such that the RNAbinding protein or RNA binding domain in the RNA delivery moleculerecognizes and is able to bind to the RNA recognition site, specific forthe RNA binding protein or RNA binding domain, in the chimeric RNAimolecule.

In one specific embodiment, the invention relates to a complex betweenan RNA delivery molecule and a chimeric RNAi molecule, wherein theRNA-binding domain of the RNA delivery molecule comprises the coliphageHK022 Nun protein or a fragment thereof, said fragment comprising theRNA-binding domain of the HK022 Nun protein, and wherein the chimericRNAi molecule comprising the double stranded RNA molecule furthercomprises a nucleotide sequence corresponding to the binding motif forbinding to the coliphage HK022 Nun protein or the fragment thereof. In apreferred embodiment, the RNA-binding domain is a polypeptide comprisingthe sequence represented in any of SEQ ID NOs 2, 4, 6, 8 or 87, mostpreferably comprising the sequence represented in SEQ ID NO 8, and thechimeric RNAi molecule comprising the double stranded RNA furthercomprises the binding motif represented in SEQ ID NO 15 or 16. Inanother preferred embodiment, the RNA-binding domain is a polypeptidecomprising the sequence represented in any of SEQ ID NOs 2, 4, 6, 8 or87, most preferably comprising the sequence represented in SEQ ID NO 8,and the chimeric RNAi molecule is any of the chimeric RNAi molecules asrepresented by any of SEQ ID NOs 45 to 48, 55, 61 to 64, 70, 76 to 79,and 85.

In another preferred embodiment, the invention relates to a complexbetween an RNA delivery molecule and a chimeric RNAi molecule whereinthe RNA-binding domain of the RNA delivery molecule is the Bacillussubtilis LicT protein or a fragment thereof, said fragment comprisingthe RNA binding domain and wherein the chimeric RNAi molecule comprisingthe double stranded RNA molecule comprises a nucleotide sequencecorresponding to the binding motif for binding to the Bacillus subtilisLicT protein or the fragment thereof. In a preferred embodiment the RNAbinding domain is a polypeptide comprising the sequence represented inSEQ ID NO 10 or 12, preferably a polypeptide comprising the sequencerepresented in SEQ ID NO 12, and the chimeric RNAi molecule comprisingthe double stranded RNA molecule comprises the binding motif representedin FIG. 41, or as represented in any of SEQ ID NOs 17 to 34. In anotherpreferred embodiment, the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 10 or 12, preferably a polypeptidecomprising the sequence represented in SEQ ID NO 12, and the chimericRNAi molecule is any of the chimeric RNAi molecules as represented byany of SEQ ID NOs 41 to 44, 54, 57 to 60, 69, 72 to 75, and 84.

In yet another preferred embodiment, the invention relates to a complexbetween an RNA delivery molecule and a chimeric RNAi molecule whereinthe RNA-binding domain of the RNA delivery molecule is MS2 or a fragmentthereof, said fragment comprising the RNA binding domain and wherein thechimeric RNAi molecule comprising the double stranded RNA moleculecomprises a nucleotide sequence corresponding to the binding motif forbinding to MS2 or the fragment thereof. In a preferred embodiment, theRNA binding domain is a polypeptide comprising the sequence representedin SEQ ID NO 14 and the chimeric RNAi molecule comprising the doublestranded RNA molecule comprises the binding motif represented in FIG.42, or as represented in any of SEQ ID NOs 35 to 40. In anotherpreferred embodiment, the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 14, and the chimeric RNAi moleculeis any of the chimeric RNAi molecules as represented by any of SEQ IDNOs 49 to 52, 56, 65 to 68, 71, 81 to 83, and 86.

Table 5 provides an overview of specific embodiments of complexesaccording to the invention comprising an RNA delivery molecule showing aspecific RNA binding domain, combined with a chimeric RNAi moleculecomprising RNA sequences that are recognized by said specific RNAbinding domain of the RNA delivery molecule as indicated in Table 5.

TABLE 5 RNA binding domain RNA sequences recognized of the RNA deliverymolecule by RNA binding domain SEQ ID NOs 2, 4, 6 or 8 SEQ ID NO 15 and16 (coliphage HK022 Nun protein (*)) SEQ ID NO 10 or 12 SEQ ID NOs 17 to34 (Bacillus subtilis LicT protein (*)) SEQ ID NO 14 SEQ ID NOs 35 to 40(bacteriophage MS2 coat protein (*)) SEQ ID NO 87 SEQ ID NO 15 and 16(N-terminal domain of Bacteriophage λ N protein) (*) or essential parts,or homologues thereof

Table 6 provides an overview of other specific embodiments of complexesaccording to the invention comprising an RNA delivery moleculecontaining an RNA binding domain, combined with a specific chimeric RNAimolecule as indicated in Table 6.

TABLE 6 RNA binding domain of the RNA delivery molecule chimeric RNAimolecule amino acid sequences nucleic acid sequences SEQ ID NOs 2, 4, 6or 8 SEQ ID NOs 45, 46, 47, 48, 55, 61, 62, 63, 64, 70, (coliphage HK022Nun protein (*)) 76, 77, 78, 79, 85 SEQ ID NO 10 or 12 SEQ ID NOs 41,42, 43, 44, 54, 57, 58, 59, 60, 69, (Bacillus subtilis LicT protein (*))72, 73, 74, 75, 84 SEQ ID NO 14 SEQ ID NOs 49, 50, 51, 52, 56, 65, 66,67, 68, 71, (bacteriophage MS2 coat protein (*)) 80, 81, 82, 83, 84, 86(*) or essential parts, or homologues thereof

It shall be understood from the present description that the present RNAdelivery molecules are particularly suitable for improving the deliveryof dsRNA to pest organisms which are feeding on a plant expressing dsRNAfor the purpose of eliciting RNA interference in the pest organism.

In one embodiment the present invention relates to an RNA deliverymolecule comprising a fusion protein which consists of an RNA bindingdomain fused, preferably by means of a linker polypeptide, to atargeting module. Preferably said targeting module comprises a domainthat is endocytosed in the gut of a target organism. The present RNAdelivery molecule is capable of forming a complex with the chimeric RNAimolecule comprising dsRNA for the purpose of interference.

In another embodiment the present invention relates to an RNA deliverymolecule comprising a fusion protein which consists of an RNA bindingdomain fused preferably by means of a linker polypeptide, to a domainthat is transcytosed from the gut to the haemolymph or coelumic fluid ofa target organism. The present RNA delivery molecule is capable offorming a complex with the chimeric RNAi molecule comprising dsRNA forthe purpose of interference.

F. Methods

The present invention relates to methods for delivering dsRNA to a pestspecies, to methods for down-regulating the expression of a target genein a pest species and to methods for producing transgenic plantsresistant to pest species.

Methods according to the present invention include feeding the RNAdelivery molecules to the organism to deliver the dsRNA to the organismtissues. It is envisaged that the methods of the invention will have usein controlling plant diseases caused by feeding organisms. Methods ofpest control of organisms, and of protecting plants against organismsare provided.

The RNA delivery molecule and the dsRNA for purposes of RNA interferencemay be taken up by the pest organism in several ways.

The invention relates to methods for delivering one or moredouble-stranded RNA molecules to a pest species, comprising:

-   -   co-expressing in a plant cell of:        -   a) an RNA delivery molecule according to the invention, and        -   b) an RNA molecule which comprises double-stranded RNA            comprising annealed complementary strands, one of which has            a nucleotide sequence which is complementary to at least            part of a target nucleotide sequence of a target gene of the            pest species, and which further comprises a nucleotide            sequence which binds to the RNA-binding domain of the RNA            delivery molecule of (a), and    -   feeding said plant cell to said pest species.

In another embodiment, a method is provided for deliveringdouble-stranded RNA to a pest species, comprising feeding the pestspecies with a mixture of an RNA molecule and an RNA delivery moleculeaccording to the present invention,

-   -   wherein the RNA molecule comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene of the pest species,        and further comprises a nucleotide sequence which binds to the        RNA-binding domain of the RNA delivery molecule,    -   whereby the mixture is taken up into the gut of the pest        species, and    -   whereby the complex of the double-stranded RNA bound to the RNA        delivery molecule is transcytosed and/or endocytosed by a gut        cell and/or a tissue cell.

In one embodiment the invention also relates to a method fordown-regulating expression of a target gene in a pest species,comprising feeding the pest species with a mixture of an RNA moleculeand an RNA delivery molecule according to the present invention,

-   -   wherein the RNA molecule comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene to be        down-regulated, and further comprises a sequence which binds to        the RNA-binding domain of the RNA delivery molecule,    -   whereby the mixture is taken up into the gut of the pest        species, and    -   whereby the complex of the double-stranded RNA bound to the RNA        delivery molecule is transcytosed and/or endocytosed by a gut        cell and/or a tissue cell and thereby down-regulates expression        of the target gene in a pest cell.

In yet another embodiment, a method is provided for deliveringdouble-stranded RNA to a pest species, comprising feeding the pestspecies with a complex comprising an RNA delivery molecule as definedherein, and at least one chimeric RNAi molecule consisting of an RNAmolecule wherein the RNA molecule comprises double-stranded RNAcomprising annealed complementary strands, one of which has a nucleotidesequence which is complementary to at least part of a target nucleotidesequence of the target gene to be down-regulated, and further comprisesa sequence which binds to the RNA-binding domain of the RNA deliverymolecule,

-   -   whereby the complex is taken up into the gut of the pest        species, and    -   whereby the complex of the double-stranded RNA bound to the RNA        delivery molecule is transcytosed and/or endocytosed by a gut        cell and/or a tissue cell.

The term “feeding” according to the present invention may refer tofeeding of the pest species with a host cell or organism, e.g. plantcell, bacteria, fungi, yeast, etc., or a mixture thereof comprising orexpressing, sprayed with or coated with at least one RNA deliverymolecule and/or chimeric RNAi molecule of the invention. In oneembodiment, a mixture of bacteria or plant(s) cell(s) may be fed to apest species, whereby the mixture comprises at least one bacterium orplant (cell) that comprises or expresses an RNA delivery molecule and atleast another bacterium or plant (cell) that comprises or expresses achimeric RNAi molecule.

In yet another embodiment, the invention relates to a method fordown-regulating expression of a target gene in a pest species,comprising:

-   -   co-expressing in a plant cell of:    -   (a) an RNA delivery molecule according to the invention,    -   (b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene to be        down-regulated, and which further comprises a nucleotide        sequence which binds to the RNA-binding domain of the RNA        delivery molecule of (a), and    -   feeding said plant cell to said pest species.

In more specific embodiments, the invention relates to any of themethods described above wherein the RNA-binding domain of an RNAdelivery molecule (a) comprises the coliphage HK022 Nun protein or afragment thereof, said fragment comprising the RNA-binding domain of theHK022 Nun protein, and wherein the double stranded RNA molecule (b)comprises a nucleotide sequence corresponding to the binding motif forbinding to the coliphage HK022 Nun protein or the fragment thereof. Evenmore specific, the invention relates to such a method wherein theRNA-binding domain is a polypeptide comprising the sequence representedin any of SEQ ID NOs 2, 4, 6, 8 or 87, preferably comprising thesequence represented in SEQ ID NO 8, and wherein the double stranded RNAmolecule (b) comprises the binding motif represented in SEQ ID NO 15 or16.

In other specific embodiments, the invention relates to any of themethods described above wherein in (a) the RNA binding domain is theBacillus subtilis LicT protein or a fragment thereof, said fragmentcomprising the RNA binding domain and wherein the double stranded RNAmolecule (b) comprises a nucleotide sequence corresponding to thebinding motif for binding to the Bacillus subtilis LicT protein or thefragment thereof. Even more specific, the invention relates to such amethod wherein the RNA binding domain is a polypeptide comprising thesequence represented in SEQ ID NO 10 or 12, preferably a polypeptidecomprising the sequence represented in SEQ ID NO 12, and wherein thedouble stranded RNA molecule (b) comprises the binding motif representedin FIG. 41, or as represented in any of SEQ ID NOs 17 to 34.

In other specific embodiments, the invention relates to any of themethods described above wherein in (a) the RNA binding polypeptide isMS2 or a fragment thereof said fragment comprising the RNA bindingdomain and double stranded RNA molecule (b) comprises a nucleotidesequence corresponding with the binding motif for binding to MS2 or thefragment thereof. Even more specific, the invention relates to such amethod wherein in (a) the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 14, and wherein (b) comprises thebinding motif represented in FIG. 42 or as represented in any of SEQ IDNOs 35 to 40.

A method for producing a transgenic plant that is resistant to a pestspecies, comprising:

-   -   co-expressing in a plant cell or plant tissue of:    -   (a) an RNA delivery molecule according to the present invention,        and    -   (b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene of said pest        species, and which further comprises a nucleotide sequence which        binds to the RNA-binding domain of the RNA delivery molecule of        (a), and    -   regenerating a plant from said plant cell or plant tissue.

In more specific embodiments, the invention relates to any of themethods described above for producing a transgenic plant that isresistant to a pest species, wherein the RNA-binding domain of an RNAdelivery molecule (a) comprises the coliphage HK022 Nun protein or afragment thereof, said fragment comprising the RNA-binding domain of theHK022 Nun protein, and wherein the double stranded RNA molecule (b)comprises a nucleotide sequence corresponding to the binding motif forbinding to the coliphage HK022 Nun protein or the fragment thereof. Evenmore specific, the invention relates to such a method wherein theRNA-binding domain is a polypeptide comprising the sequence representedin any of SEQ ID NOs 2, 4, 6, 8 or 87, preferably comprising thesequence represented in SEQ ID NO 8, and wherein the double stranded RNAmolecule (b) comprises the binding motif represented in SEQ ID NO 15 or16.

In other specific embodiments, the invention relates to any of themethods described above for producing a transgenic plant that isresistant to a pest species wherein in (a) the RNA binding domain is theBacillus subtilis LicT protein or a fragment thereof, said fragmentcomprising the RNA binding domain and wherein the double stranded RNAmolecule (b) comprises a nucleotide sequence corresponding to thebinding motif for binding to the Bacillus subtilis LicT protein or thefragment thereof. Even more specific, the invention relates to such amethod wherein the RNA binding domain is a polypeptide comprising thesequence represented in SEQ ID NO 10 or 12, preferably a polypeptidecomprising the sequence represented in SEQ ID NO 12, and wherein thedouble stranded RNA molecule (b) comprises the binding motif representedin FIG. 41, or as represented in any of SEQ ID NOs 17 to 34.

In other specific embodiments, the invention relates to any of themethods described above for producing a transgenic plant that isresistant to a pest species wherein in (a) the RNA binding polypeptideis MS2 or a fragment thereof said fragment comprising the RNA bindingdomain and double stranded RNA molecule (b) comprises a nucleotidesequence corresponding with the binding motif for binding to MS2 or thefragment thereof. Even more specific, the invention relates to such amethod wherein in (a) the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 14, and wherein (b) comprises thebinding motif represented in FIG. 42 or as represented in any of SEQ IDNOs 35 to 40.

In yet another embodiment, the method for producing a transgenic plantthat is resistant to a pest species, comprises:

-   -   co-expressing in a plant cell or plant tissue of:    -   a) an RNA delivery molecule according to the present invention,        and    -   b) a chimeric RNAi molecule according to the present invention        and preferably comprising    -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, for instance as represented in        any of SEQ ID NOs 15 to 40, or an RNA secondary structure as        represented in FIG. 41 or 42, and    -   at least one nucleotide sequence corresponding to a target        nucleotide sequence of a target gene of a pest species, and    -   regenerating a plant from said plant cell or plant tissue.

Even more specific, the invention relates to such a method for producinga transgenic plant that is resistant to a pest species wherein saidchimeric RNAi molecule is a molecule as described in Example 2.According to further specific embodiments, the invention relates to sucha method for producing a transgenic plant that is resistant to a pestspecies wherein said chimeric RNAi molecule has a sequence asrepresented any of FIGS. 43 to 54 (any of SEQ ID NOs 41 to 52) or FIGS.56 to 58 (any of SEQ ID NOs 54 to 56), or any of FIGS. 60 to 74 (any ofSEQ ID NOs 57 to 71), or any of FIGS. 76 to 90 (any of SEQ ID NOs 72 to86). In this context it should be understood that the chimeric RNAimolecule is recognized and able to be bound by the RNA binding proteinor RNA binding domain present in the RNA delivery molecule according tothe invention.

Thus, the present invention provides for methods for the production oftransgenic plants, plant cells or plant tissues comprising theintroduction of a nucleic acid or vector according to the invention intothe genome of said plant, plant cell or plant tissue.

The present invention also relates to a transgenic plant resistant to apest species, an essential derived variety thereof, plant part, plantcell or protoplast thereof obtainable by any of the methods as describedherein.

In another embodiment, the invention relates to a transgenic plant,essentially derived variety thereof, plant part, plant cell orprotoplast thereof which comprises a nucleic acid encoding an RNAdelivery molecule as defined herein, wherein said nucleic acid isheterologous to the genome of said transgenic plant, or an essentiallyderived variety thereof, plant part, plant cell or plant protoplastthereof.

The invention also relates to a transgenic plant, essentially derivedvariety thereof, plant part, plant cell or protoplast thereof whichcomprises a nucleic acid encoding an RNA delivery molecule as definedherein, and which comprises a nucleic acid encoding a chimeric RNAimolecule as defined herein, wherein said nucleic acids are heterologousto the genome of said transgenic plant, or an essentially derivedvariety thereof, plant part, plant cell or plant protoplast thereof.

In yet another embodiment, the invention provides a transgenic plantwhich comprises a vector as described herein.

As used herein, ‘transgenic plant’ includes reference to a plant, whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a vector. ‘Transgenic’ is used herein to include any cell,cell line, callus, tissue, plant part or plant, the genotype of whichhas been altered by the presence of the heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic.

In another embodiment of the invention, there is provided a plant,essentially derived variety thereof, plant part, plant cell orprotoplast thereof wherein the plant, essentially derived varietythereof, plant part, plant cell, or protoplast thereof has beentransformed with a nucleic acid encoding an RNA delivery molecule asdefined herein, and optionally also with a nucleic acid encoding acorresponding chimeric RNAi molecule as described herein.

The present invention also provides a plant, essentially derived varietythereof, plant part, plant cell, or protoplast thereof whichco-expresses:

-   -   a) an RNA delivery molecule as described herein, and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene in a pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a).

In another embodiment of the invention also provides a plant,essentially derived variety thereof, plant part, plant cell, orprotoplast thereof which co-expresses:

-   -   a) an RNA delivery molecule as defined herein, and    -   b) a chimeric RNAi molecule according to the present invention        and preferably comprising    -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, for instance as represented in        any of SEQ ID NOs 15 to 40, or an RNA secondary structure as        represented in FIG. 41 or 42, and    -   at least one nucleotide sequence corresponding to a target        nucleotide sequence of a target gene of a pest species.

Even more specific, the invention relates to a plant, essentiallyderived variety thereof, plant part, plant cell, or protoplast thereofwhich co-expresses an RNA delivery molecule as defined herein, and achimeric RNAi molecule according to the present invention, wherein saidchimeric RNAi molecule has a sequence as represented any of FIGS. 43 to54 (any of SEQ ID NOs 41 to 52) or FIGS. 56 to 58 (any of SEQ ID NOs 54to 56), or any of FIGS. 60 to 74 (any of SEQ ID NOs 57 to 71), or any ofFIGS. 76 to 90 (any of SEQ ID NOs 72 to 86). In this context it shouldbe understood that the chimeric RNAi molecule is recognized and able tobe bound by the RNA binding protein or RNA binding domain present in theRNA delivery molecule according to the invention.

In one specific embodiment, the invention relates to a plant,essentially derived variety thereof, plant part, plant cell, orprotoplast thereof which co-expresses an RNA delivery molecule asdefined herein, and a chimeric RNAi molecule according to the presentinvention, wherein the RNA-binding domain of the RNA delivery moleculecomprises the coliphage HK022 Nun protein or a fragment thereof, saidfragment comprising the RNA-binding domain of the HK022 Nun protein, andwherein the chimeric RNAi molecule comprises a nucleotide sequencecorresponding to the binding motif for binding to the coliphage HK022Nun protein or the fragment thereof. In a preferred embodiment, theRNA-binding domain is a polypeptide comprising the sequence representedin any of SEQ ID NOs 2, 4, 6, 8 or 87, most preferably comprising thesequence represented in SEQ ID NO 8, and the chimeric RNAi moleculecomprises the binding motif represented in SEQ ID NO 15 or 16. Inanother preferred embodiment, the RNA-binding domain is a polypeptidecomprising the sequence represented in any of SEQ ID NOs 2, 4, 6, 8 or87 most preferably comprising the sequence represented in SEQ ID NO 8,and the chimeric RNAi molecule is any of the chimeric RNAi molecules asrepresented by any of SEQ ID NOs 45 to 48, 55, 61 to 64, 70, 76 to 79,and 85.

In another specific embodiment, the invention relates to a plant,essentially derived variety thereof, plant part, plant cell, orprotoplast thereof which co-expresses an RNA delivery molecule asdefined herein, and a chimeric RNAi molecule according to the presentinvention, wherein the RNA-binding domain of the RNA delivery moleculeis the Bacillus subtilis LicT protein or a fragment thereof, saidfragment comprising the RNA binding domain and wherein the chimeric RNAimolecule le comprises a nucleotide sequence corresponding to the bindingmotif for binding to the Bacillus subtilis LicT protein or the fragmentthereof. In a preferred embodiment the RNA binding domain is apolypeptide comprising the sequence represented in SEQ ID NO 10 or 12,preferably a polypeptide comprising the sequence represented in SEQ IDNO 12, and the chimeric RNAi molecule comprises the binding motifrepresented in FIG. 41, or as represented in any of SEQ ID NOs 17 to 34.In another preferred embodiment, the RNA binding domain is a polypeptidecomprising the sequence represented in SEQ ID NO 10 or 12, preferably apolypeptide comprising the sequence represented in SEQ ID NO 12, and thechimeric RNAi molecule is any of the chimeric RNAi molecules asrepresented by any of SEQ ID NOs 41 to 44, 54, 57 to 60, 69, 72 to 75and 84.

In yet another specific embodiment, the invention relates to a plant,essentially derived variety thereof, plant part, plant cell, orprotoplast thereof which co-expresses an RNA delivery molecule asdefined herein, and a chimeric RNAi molecule according to the presentinvention, wherein the RNA-binding domain of the RNA delivery moleculeis MS2 or a fragment thereof, said fragment comprising the RNA bindingdomain and wherein the chimeric RNAi molecule comprises a nucleotidesequence corresponding to the binding motif for binding to MS2 or thefragment thereof. In a preferred embodiment, the RNA binding domain is apolypeptide comprising the sequence represented in SEQ ID NO 14 and thechimeric RNAi molecule comprises the binding motif represented in FIG.42, or as represented in any of SEQ ID NOs 35 to 40. In anotherpreferred embodiment, the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 14, and the chimeric RNAi moleculeis any of the chimeric RNAi molecules as represented by any of SEQ IDNOs 49 to 52, 56, 65 to 68, 71, 80 to 83 and 86.

The term “transformation” as used herein, refers to the transfer of anexogenous polynucleotide into a host cell, irrespective of the methodused for the transfer. The polynucleotide may be transiently or stablyintroduced into the host cell or organism and may be maintainednon-integrated, for example, as a plasmid, or alternatively, may beintegrated into the host genome. Transformation may be transient orstable. The invention thus also relates to such a stably or transientlytransformed transgenic plant, plant cell or plant tissue. The inventionfurther relates to any plant which comprises any of the subject vectorsin accordance with the invention.

According to a further embodiment, the invention also relates to any ofthe transgenic plants described herein comprising a nucleic acidencoding an RNA delivery molecule as defined herein characterized inthat said plant has increased resistance to a pest organism, forinstance increased resistance of between 30% to 80% compared to controlplants.

In other embodiments, the invention also relates to the progeny of aplant or essentially derived variety thereof obtainable by a method ofthe present invention or as described herein which has been obtained ina generative or vegetative manner.

The present invention also includes parts or derivatives of obtainableby a method of the present invention or as described herein, such as butnot limited to leaves, stems, roots, shoots, cuttings or explants andthe like, protoplasts, somatic embryos, anthers, petioles, cells inculture, seeds, flowers, fruits and tubers.

In another embodiment the invention relates to a method for controllinga pest species comprising feeding said pest species with a mixture of anRNA molecule and an RNA delivery molecule as defined herein,

-   -   wherein the RNA molecule comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene of said pest        species and further comprises a nucleotide sequence which binds        to the RNA-binding domain of the RNA delivery molecule,    -   whereby the mixture is taken up into the gut of the pest        species, and    -   whereby a complex is formed between the double-stranded RNA of        said RNA molecule and said RNA delivery molecule,    -   whereby the complex of the double-stranded RNA bound to the RNA        delivery molecule is transcytosed and/or endocytosed by a gut        cell and/or a tissue cell; and    -   whereby the double-stranded RNA causes RNAi interference with        the target gene in a pest cell such that the pest species is        killed or paralyzed.

In yet another embodiment the invention relates to a method forcontrolling a pest species comprising feeding the pest species with acomplex comprising an RNA delivery molecule as defined herein, and atleast one chimeric RNAi molecule consisting of an RNA molecule whereinthe RNA molecule comprises double-stranded RNA comprising annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to at least part of a target nucleotide sequence of thetarget gene to be down-regulated, and further comprises a sequence whichbinds to the RNA-binding domain of the RNA delivery molecule,

-   -   whereby the complex is taken up into the gut of the pest        species, and    -   whereby the complex of the double-stranded RNA bound to the RNA        delivery molecule is transcytosed and/or endocytosed by a gut        cell and/or a tissue cell, and    -   whereby the double-stranded RNA causes RNAi interference with        the target gene in a pest cell such that the pest species is        killed or paralyzed.

In yet another embodiment the invention also relates to a method forcontrolling a pest species comprising feeding the pest species with atransgenic plant or any progeny or part thereof as defined herein whichis resistant to said pest species.

In yet another embodiment a method is provided for protecting a plantagainst a pest organism comprising co-expressing in said plant of:

-   -   a) an RNA delivery molecule as defined herein, and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of the target gene to be        down-regulated, and which further comprises a nucleotide        sequence which binds to the RNA-binding domain of the RNA        delivery molecule of (a).

In yet another embodiment a method is provided for protecting a plantagainst a pest organism comprising co-expressing in said plant:

-   -   a) an RNA delivery molecule as defined herein, and    -   b) a chimeric RNAi molecule according to the present invention        and preferably comprising        -   at least one nucleotide sequence recognized by an            RNA-binding protein or RNA-binding domain, for instance as            represented in any of SEQ ID NOs 15 to 40, or an RNA            secondary structure as represented in FIG. 41 or 42, and        -   at least one nucleotide sequence corresponding to a target            nucleotide sequence of a target gene of a pest species.

Even more specific, said chimeric RNAi molecule has a sequence asrepresented in any of FIGS. 43 to 54 (any of SEQ ID NOs 41 to 52) orFIGS. 56 to 58 (any of SEQ ID NOs 54 to 56), or in any of FIGS. 60 to 74(any of SEQ ID NOs 57 to 71), or in any of FIGS. 76 to 90 (any of SEQ IDNOs 72 to 86). In this context it should be understood that the chimericRNAi molecule is recognized and able to be bound by the RNA bindingprotein or RNA binding domain present in the RNA delivery moleculeaccording to the invention.

The invention further relates to any of the methods described hereinwherein the said pest species is any pest species described herein.

Plants

In a preferred embodiment the host organism is a plant and the pestspecies is a plant pathogenic pest.

The term “plant” as used herein encompasses any plant material such asinter alia a plant cell, plant tissue (including callus), plant part,whole plant, ancestors and progeny. A plant part may be any part ororgan of the plant and include for example a seed, fruit, stem, leaf,shoot, flower, anther, root or tuber. The plant material should express,or have the capability to express, RNA delivery molecules, chimeric RNAimolecules, and dsRNA corresponding to one or more target genes of thepest species to be killed or paralyzed. The term “plant” alsoencompasses suspension cultures, embryos, meristematic regions, callustissue, gametophytes, sporophytes, pollen, and microspores. The plant asused herein refers to all plants including algae, ferns and trees. In apreferred embodiment the plant belongs to the superfamily ofViridiplantae, further preferably is a monocot or a dicot. According toone embodiment of the present invention, the plant is susceptible toinfestation by a plant pathogenic and/or parasitic nematode, by a fungusor an insect. Particular plants useful in the methods of the presentinvention are crop plants including for example monocots such as sugarcane and cereals (including wheat, oats, barley, sorghum, rye, millet,corn, rice) and dicots such as potato, tomato, vine, apple, pear,banana, sunflower, soybean, canola, alfalfa, rapeseed and cotton.Particular trees that can be used in the methods of the presentinvention are pine, eucalyptus and poplar.

“Administering” a DNA to a cell may be achieved by a variety of means,each well known by the person skilled in the art. Examples of usefultechniques are shot-gun, ballistics, electroporation, transfection andtransformation. For particular embodiments of the present inventionwhere the cell is a plant cell, general techniques for expression ofexogenous double-stranded RNA in plants for the purposes of RNAi areknown in the art (Baulcombe D, 2004, Nature. 431(7006):356-63. RNAsilencing in plants the contents of which are incorporated herein byreference). General techniques for expression of proteins in plants areknown in the art (Fischer R et al. 1999, Biotechno. Appl. Biochem. (Pt2)99-101 and 101-108; or Cunningham C. 1997 (Ed.). Recombinant Proteinsfrom Plants, 308p). More particularly, methods for expression ofdouble-stranded RNA in plants for the purposes of down-regulating geneexpression in plant pests such as nematodes or insects are also known inthe art. Similar methods can be applied in an analogous manner in orderto express the RNA delivery molecule and/or the chimeric RNAi moleculein plants for the purposes of down-regulating expression of a targetgene in a plant pest species. In order to achieve this effect it isnecessary only for the plant to express (transcribe) the RNA deliverymolecule and/or the chimeric RNAi molecule in a part of the plant whichwill come into direct contact with the pest species, such that the RNAdelivery molecule and/or the chimeric RNAi molecule (or the complex) canbe taken up by the pest species. Depending on the nature of the pestspecies and its relationship with the host plant, expression of the RNAdelivery molecule and/or the chimeric RNAi molecule could occur within acell or tissue of a plant within which the pest species is also presentduring its life cycle, or the RNA delivery molecule and/or the chimericRNAi molecule may be secreted into a space between cells, such as theapoplast, that is occupied by the pest species during its life cycle.Furthermore, the RNA delivery molecule and/or the chimeric RNAi moleculemay be located in the plant cell, for example in the cytosol, or in theplant cell organelles such as chloroplast, mitochondrion, vacuole orendoplasmatic reticulum.

Alternatively, the RNA delivery molecule and/or the chimeric RNAimolecule may be secreted by the plant cell and by the plant to theexterior of the plant. As such, the RNA delivery molecule and/or thechimeric RNAi molecule may form a protective layer on the surface of theplant.

The present invention thus relates to a method for the production of atransgenic cell or organism, comprising the step of administering amolecule, nucleic acid or a vector as described herein to said cell ororganism. Preferably, said cell is a plant cell or said organism is aplant. The invention further relates to any transgenic cell ortransgenic organism obtainable by the above described method, preferablysaid transgenic cell or organism is plant cell or plant organism.

The methods of the present invention for the production of transgenicorganism may further comprise the steps of cultivating the transgeniccell under conditions promoting growth and development. Where thetransgenic organism is a plant, these methods may further comprise thesteps of regenerating a plant from plant tissue, allowing growth toreach maturity and to reproduce. Alternatively, the transgenic planttissue may take other forms or may form part of another plant, examplesof which are chimera plants and grafts (for example a transformedrootstock grafted to an untransformed scion). Advantapes

Uses of the present RNA delivery molecule for delivering dsRNA from aplant to a feeding organism are numerous.

A first major advantage of the delivery system according to the presentinvention is that it permits to deliver dsRNA more efficientlyintracellularly in target organisms. The present system enables thedelivery and uptake of the chimeric RNAi molecules of the invention,comprising double stranded RNA for the purpose of RNA interference. Thechimeric RNAi molecules may even comprise small RNA fragments, e.g. 21mers, and efficiently deliver these to the gut cell of a target pestorganism. Feeding a target organism dsRNA that has been bound to adelivery molecule according to the present invention and that isefficiently delivered results in an improved uptake of the dsRNA. As aresult thereof, lower amounts of dsRNA need to be used in order toobtain a suitable effect in the target organism.

In the case that the RNA delivery molecule according to the presentinvention comprises a domain that binds a protein that is endocytosed inthe gut of a target organism or that binds to an endocytosis receptor,the complex between the RNA delivery molecule and the chimeric RNAimolecule which is formed in the target organism or taken up by thetarget organism by feeding can enter the gut cells through endocytosis,e.g. by entering endocytic vesicles, and from there are capable ofentering the cell cytoplasm. In the endosome the protein-RNA complex maybe dissociated through proteolysis or pH-dependent dissociation. Afraction of the dsRNA will then enter the cell in a manner similar tothe delivery mechanism for antisense RNAs, as is well known in theliterature.

The gut of an insect is a hostile environment, having low or high pHvalues and RNases which degrade RNA. High pH such as found inlepidopteran guts degrades RNA chemically enhanced. Transcytosis of theRNA molecules to the haemolymph enables to quickly remove the dsRNA froman RNase rich and/or pH hostile environment. In the case that the RNAdelivery molecule according to the present invention comprises a domainthat binds a protein that is transcytosed through the gut of a targetorganism to the haemolymph or coelomic fluid, or that binds to atranscytosis receptor, the chimeric RNA molecules which are taken up bythe target organism by feeding can enter the gut and pass through thegut to the haemolymph or coelomic fluid. Transcytosis of the chimericRNAi molecules to the haemolymph or coelomic fluid allows to direct thedsRNA to a broad range of target tissues including muscles, CNS, andother.

In other cases, it may also be required to have a combination ofendocytosis and transcytosis to have efficient and site-specificdelivery of the dsRNA bound to the delivery molecule.

Another advantage of the present RNA delivery molecule is that whenforming a complex with the dsRNA, it effectively protects the dsRNAmolecules from degradation in the plant and in the gut of the targetorganism, which is a very hostile environment.

The present RNA delivery molecule may thus allow expression of long aswell as short dsRNA fragments in plants, to be delivered by feeding to atarget organism. Usually, long (of e.g. 80 bp or more) dsRNA fragmentsare expressed in a plant for delivery by feeding to a target organism.Expression of long dsRNA fragments involves several disadvantages. Forinstance, it makes it necessary to protect these long RNA fragments inthe plant cytoplasm from Dicer activity in the endogenous plant. Dicingof longer RNA fragments may result in diced fragments that may createdominant negative effects in plants through tittering away RISC which isneeded for normal plant growth and physiology, or which may downregulate plant genes or chromatic or even the transgenic dsRNAexpressing gene. Smaller dsRNA molecules (even as short as 21 bp) may beeffectively fed to target organisms and taken up in the gut enterocytesleading to target knockdown. As a consequence thereof, it may be notlonger required to express long RNA fragments of 80 bp or longer in theplants and shorter—and thus more specific and selective—target fragmentscan be expressed in plants.

In addition, in the case long RNA fragments of 80 bp or longer are stillto be expressed in the plants, using the present RNA delivery moleculewhich protect the RNA, may obviate the need of additionally protectingthese fragments from dicing activity in the endogenous plant.

Another major advantage of the present delivery system is that thedelivery molecule can be used for improving the delivery of chimericRNAi molecules using a sequence or structure specific RNA bindingprotein. The present RNA delivery system can advantageously be used forbinding different categories of RNA. In particular, the present systemis capable of binding dsRNA generically, or dsRNA, ssRNA or RNAstructures specifically, as long as they contain the recognitionsequence or structure. In a preferred embodiment, the present RNAdelivery system can be used to bind a chimeric RNAi consisting of atarget specific region (target dsRNA) for the purpose of RNAinterference, and an RNA sequence that specifically binds the RNAdelivery molecule, and in particular the RNA binding domain thereof.Advantageously, in such case the dsRNA would not interact with plantDicer. Use of a chimeric RNAi bound to the RNA delivery molecule of thepresent invention also allows the use of long RNA fragments in plants,which will not be processed to diced fragments that could inducenegative side-effects in the plants as explained above. In addition,binding of a chimeric RNAi molecule to an RNA delivery moleculeaccording to the present invention permits the transgenic dsRNAmolecules to be accumulated in a selected compartment in the plant, andfor instance in the nucleolus, nucleus, cytoplasm tRNA or ribosome,phloem, etc., which allows a more efficient protection, accumulation ordelivery to a target organism.

G. Uses

In general, the present invention also relates to the use of RNAdelivery molecules or complexes thereof with a chimeric RNAi moleculefor various agronomic and research applications requiring the deliveryof dsRNA into a target pest organism.

In an embodiment, the invention relates to the use of an RNA deliverymolecule as described herein for delivering dsRNA to a pest species.

In another embodiment, the invention also relates to the use of an RNAdelivery molecule in combination with a corresponding chimeric RNAimolecule as described herein for delivering dsRNA to a pest species.

It shall be understood from the present description that the present RNAdelivery molecules are particularly suitable for improving the deliveryof dsRNA to pest organisms which are feeding on a plant expressingdsRNA. Alternatively, it is apparent that the present RNA deliverymolecules according to the present invention may also be very useful forimproving the delivery of dsRNA to pest organisms by any other way,including but not limited to injection of dsRNA, soaking the organismsin dsRNA solution or by feeding the pest organisms Escherichia colibacteria that simultaneously express sense and antisense RNAs and thatcan acquire dsRNA.

In yet another embodiment, the present invention relates to the use ofRNA delivery molecules in combination with corresponding chimeric RNAimolecules for down-regulating the expression of target genes in pestspecies.

In another embodiment, the present complexes between RNA deliverymolecules and corresponding chimeric RNAi molecules are also very usefulfor down-regulating the expression of target genes in pest species. Theinvention therefore also provides for the use of said complexes asdescribed herein for down-regulating the expression of target genes inpest species.

In yet another embodiment, the invention relates to the use of an RNAdelivery molecule as described herein for producing a transgenic plant.

In yet another embodiment, the present invention relates to the use ofan RNA delivery molecule in combination with a chimeric RNAi moleculefor producing transgenic plants resistant to pest organisms.

In another embodiment, the present complexes between an RNA deliverymolecule and a corresponding chimeric RNAi molecule are also very usefulfor producing transgenic plants which are resistant to a pest species.The invention therefore also provides for the use of said complexes asdescribed herein for producing transgenic plants resistant to pestorganisms.

In more specific embodiments, the present invention relates to the useof a transgenic plant resistant to a pest organism as described hereinfor controlling pest population growth, and/or for reducing infestationby a pest species and/or for killing or paralyzing a pest organism,and/or for preventing or reducing the amount and the number of chemical(e.g. pesticide, fungicide, nematicide) applications, and/or forreducing the environmental impact of chemical applications pesticidesand/or for reducing disease incidence in a crop and/or for improvingcrop yield.

In another embodiment the invention relates to the use of a plant,essentially derived variety thereof, plant part, plant cell orprotoplast thereof that has been transformed with (i) a nucleic acidencoding an RNA delivery molecule and (ii) a nucleic acid encoding acorresponding chimeric RNAi molecule as herein described for improvingresistance to a pest organism, and/or for controlling pest populationgrowth, and/or for preventing or reducing infestation by a pest speciesand/or for killing or paralyzing a pest organism, and/or for reducingthe amount and the number of chemical (e.g. pesticide, fungicide,nematicide) applications, and/or for reducing the environmental impactof chemical applications pesticides and/or for reducing diseaseincidence in a crop and/or for improving crop yield.

In yet another embodiment the invention relates to the use of a plant,essentially derived variety, plant part, plant cell or protoplastthereof which co-expresses:

-   -   a) an RNA delivery molecule as described herein, and    -   b) an RNA molecule which comprises double-stranded RNA        comprising annealed complementary strands, one of which has a        nucleotide sequence which is complementary to at least part of a        target nucleotide sequence of a target gene of a pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a),        for improving resistance to pest organism, and/or for        controlling pest population growth, and/or for preventing or        reducing infestation by a pest species and/or for killing or        paralyzing a pest organism, and/or for reducing the amount and        the number of chemical (e.g. pesticide, fungicide, nematicide)        applications, and/or for reducing the environmental impact of        chemical applications pesticides and/or for reducing disease        incidence in a crop and/or for improving crop yield.

In one specific embodiment, the invention relates to any of the uses asdescribed above, wherein

-   -   the RNA-binding domain of the RNA delivery molecule comprises        the coliphage HK022 Nun protein or a fragment thereof, said        fragment comprising the RNA-binding domain of the HK022 Nun        protein, preferably the RNA binding domain is a polypeptide        comprising the sequence represented in any of SEQ ID NOs 2, 4,        6, 8 or 87, most preferably comprising the sequence represented        in SEQ ID NO 8, and    -   the RNA molecule which comprises the double stranded RNA        molecule and which further comprises a nucleotide sequence        corresponding to the binding motif for binding to the coliphage        HK022 Nun protein or the fragment thereof, preferably the        binding motif represented in SEQ ID NO 15 or 16, or    -   more preferably the RNA molecule is any of the chimeric RNAi        molecules as represented by any of SEQ ID NOs 45 to 48, 55, 61        to 64, 70, 76 to 79 and 85.    -   In another specific embodiment, the invention relates to any of        the uses as described above, wherein    -   the RNA-binding domain of the RNA delivery molecule is the        Bacillus subtilis LicT protein or a fragment thereof, said        fragment comprising the RNA binding domain, preferably the RNA        binding domain is a polypeptide comprising the sequence        represented in any of SEQ ID NO 10 or 12, most preferably        comprising the sequence represented in SEQ ID NO 12, and    -   the RNA molecule which comprises the double stranded RNA        molecule comprises a nucleotide sequence corresponding to the        binding motif for binding to the Bacillus subtilis LicT protein        or the fragment thereof, preferably the binding motif        represented in FIG. 41, or as represented in any of SEQ ID NOs        17 to 34, or    -   more preferably the RNA molecule is any of the chimeric RNAi        molecules as represented by any of SEQ ID NOs 41 to 44, 54, 57        to 60, 69, 72 to 75, and 84.

In yet another specific embodiment, the invention relates to any of theuses as described above, wherein

-   -   the RNA-binding domain of the RNA delivery molecule is MS2 or a        fragment thereof, said fragment comprising the RNA binding        domain, and preferably the RNA binding domain is a polypeptide        comprising the sequence represented in SEQ ID NO 14, and    -   the RNA molecule which comprises the double stranded RNA        molecule comprises a nucleotide sequence corresponding to the        binding motif for binding to MS2 or the fragment thereof, and        preferably the binding motif represented in FIG. 42, or as        represented in any of SEQ ID NOs 35 to 40, or    -   the RNA molecule is any of the chimeric RNAi molecules as        represented by any of SEQ ID NOs 49 to 52, 56, 65 to 68, 71, 80        to 83 and 86.

In yet another embodiment, the invention relates to the use of progenyor parts or derivatives of plants obtainable from any plant oressentially derived variety thereof as described herein for improvingresistance to pest organism, and/or for controlling pest populationgrowth, and/or for preventing or reducing infestation by a pest speciesand/or for killing or paralyzing a pest organism, and/or for reducingthe amount and the number of chemical (e.g. pesticide, fungicide,nematicide) applications, and/or for reducing the environmental impactof chemical applications pesticides and/or for reducing diseaseincidence in a crop and/or for improving crop yield.

In one other specific embodiment, the method of the invention may alsobe used as a tool for experimental research, particularly in the fieldof functional genomics. Targeted down-regulation of pest genes by RNAican be used in in vitro or in vivo assays in order to study genefunction. Assays based on targeted down-regulation of specific pestgenes, leading to a measurable phenotype may also form the basis ofcompound screens for novel pesticides.

H. Compositions and Kits

In a further aspect the invention relates to a composition for reducingpest population growth and/or for killing or paralyzing a pest organismand/or for improving plant resistance to pest organisms, for improvingresistance to pest organism, and/or for controlling pest populationgrowth, and/or for preventing or reducing infestation by a pest speciesand/or for killing or paralyzing a pest organism, for reducing diseaseincidence in a crop and/or for improving crop yield, said compositioncomprising at least one RNA delivery molecule as defined herein, andmore preferably comprising at least one RNA delivery molecule and/or atleast one chimeric RNAi molecule as herein described.

According to one embodiment, the invention relates to a compositioncomprising at least one RNA delivery molecule as defined herein, andmore preferably comprising at least one RNA delivery molecule and/or atleast one chimeric RNAi molecule as herein described and a physiologicalor agronomical acceptable carrier, excipient or diluent. The inventionalso encompasses the use of said composition as a pesticide for a plantor for propagation or reproductive material of a plant.

According to yet another embodiment, the invention relates to acomposition comprising at least one RNA delivery molecule as definedherein, and more preferably comprising at least one RNA deliverymolecule and/or at least one chimeric RNAi molecule as herein described,and a physiological or agronomical acceptable carrier, excipient ordiluent.

The composition may contain further components which serve to stabilisethe dsRNA and/or prevent degradation of the dsRNA during prolongedstorage of the composition.

The composition may still further contain components which enhance orpromote uptake of the RNA delivery molecule and/or the chimeric RNAimolecule by the pest organism. These may include, for example, chemicalagents which generally promote the uptake of RNA into cells e.g.lipofectamin etc., and enzymes or chemical agents capable of digestingthe fungal cell wall, e.g. a chitinase.

The composition may be in any suitable physical form for application tothe pest, to substrates, to cells (e.g. plant cells), or to organisminfected by or susceptible to infection by a pest species. In anotherembodiment, the invention provides a composition comprising:

-   -   (a) at least one RNA delivery molecule according to the present        invention, and    -   (b) at least one RNA molecule which comprises double-stranded        RNA comprising annealed complementary strands, one of which has        a nucleotide sequence which is complementary to at least part of        a target nucleotide sequence of a target gene of a pest species,        and which further comprises a nucleotide sequence which binds to        the RNA-binding domain of the RNA delivery molecule of (a)        provided that the RNA delivery molecule is recognized and may        bind to the RNA binding protein or RNA binding domain of the RNA        delivery molecule.

The RNA molecule contained in said compositions may be (partially)self-complementary or comprise a sense and antisense strand forming adouble stranded portion.

In another embodiment, the invention provides a composition comprising:

-   -   at least one RNA delivery molecule according to the present        invention, and    -   at least one chimeric RNAi molecule comprising    -   at least a double-stranded RNA comprising annealed complementary        strands, one of which has a nucleotide sequence which is        complementary to at least part of a target nucleotide of a        target gene of said pest species (i.e. capable of causing RNAi        interference) and    -   a sequence recognized by an RNA binding protein or RNA binding        domain, for instance as represented in any of SEQ ID NOs 15 to        40, or as represented in FIG. 41 or 42.

In yet another embodiment, the present invention relates to acomposition comprising:

-   -   an RNA delivery molecule as defined herein, and    -   at least one chimeric RNAi molecule as represented by any of SEQ        ID NOs 41 to 52 or any of SEQ ID NOs 54 to 56, or any of SEQ ID        NOs 57 to 71, or any of SEQ ID NOs 72 to 86.

In one specific embodiment, the invention relates to a composition, nespecific embodiment, the invention relates to any of the uses asdescribed above, wherein

-   -   the RNA-binding domain of the RNA delivery molecule comprises        the coliphage

HK022 Nun protein or a fragment thereof, said fragment comprising theRNA-binding domain of the HK022 Nun protein, preferably the RNA bindingdomain is a polypeptide comprising the sequence represented in any ofSEQ ID NOs 2, 4, 6, 8 or 87, most preferably comprising the sequencerepresented in SEQ ID NO 8, and

-   -   the RNA molecule which comprises the double stranded RNA        molecule and which further comprises a nucleotide sequence        corresponding to the binding motif for binding to the coliphage        HK022 Nun protein or the fragment thereof, preferably the        binding motif represented in SEQ ID NO 15 or 16, or    -   more preferably the RNA molecule is any of the chimeric RNAi        molecules as represented by any of SEQ ID NOs 45 to 48, 55, 61        to 64, 70, 76 to 79 and 85.

In another specific embodiment, the invention relates to a composition,wherein

-   -   the RNA-binding domain of the RNA delivery molecule is the        Bacillus subtilis LicT protein or a fragment thereof, said        fragment comprising the RNA binding domain, preferably the RNA        binding domain is a polypeptide comprising the sequence        represented in any of SEQ ID NO 10 or 12, most preferably        comprising the sequence represented in SEQ ID NO 12, and    -   the RNA molecule which comprises the double stranded RNA        molecule comprises a nucleotide sequence corresponding to the        binding motif for binding to the Bacillus subtilis LicT protein        or the fragment thereof, preferably the binding motif        represented in FIG. 41, or as represented in any of SEQ ID NOs        17 to 34, or more preferably the RNA molecule is any of the        chimeric RNAi molecules as represented by any of SEQ ID NOs 41        to 44, 54, 57 to 60, 69, 72 to 75, and 84.

In yet another specific embodiment, the invention relates to acomposition, wherein

-   -   the RNA-binding domain of the RNA delivery molecule is MS2 or a        fragment thereof, said fragment comprising the RNA binding        domain, and preferably the RNA binding domain is a polypeptide        comprising the sequence represented in SEQ ID NO 14, and    -   the RNA molecule which comprises the double stranded RNA        molecule comprises a nucleotide sequence corresponding to the        binding motif for binding to MS2 or the fragment thereof, and        preferably the binding motif represented in FIG. 42, or as        represented in any of SEQ ID NOs 35 to 40, or    -   the RNA molecule is any of the chimeric RNAi molecules as        represented by any of SEQ ID NOs 49 to 52, 56, 65 to 68, 71, 80        to 83 and 86.

In addition, the present invention also relates to methods for producingan RNA delivery molecule described herein comprising:

-   -   (a) introducing into a host cell an isolated DNA molecule        encoding any of the RNA delivery molecules of the invention, an        nucleic acid encoding any of the RNA delivery molecules of the        invention or a vector comprising said nucleic acid;    -   (b) growing the host cell under conditions suitable for        expression of the RNA delivery molecule; and    -   (c) isolating the RNA delivery molecule produced by the host        cell.

In a preferred embodiment the present invention relates to a method forproducing an RNA delivery molecule, wherein the RNA binding domain ofsaid delivery molecule comprises a polypeptide comprising the coliphageHK022 Nun protein (for instance as represented in SEQ ID NO 2), ahomologue thereof, or a fragment thereof comprising the RNA-bindingdomain, said fragment preferably comprising amino acids 1 to 47 (SEQ IDNO 4) of the aminoterminal sequence, more preferably comprising aminoacids 13 to 47 (SEQ ID NO 6) of the aminoterminal sequence, mostpreferably comprising amino acids 22 to 47 (SEQ ID NO 8) of theaminoterminal sequence of the HK022 Nun protein.

In another preferred embodiment the present invention relates to amethod for producing an RNA delivery molecule, wherein the RNA bindingdomain of said delivery molecule comprises a polypeptide comprising theBacillus subtilis LicT protein (for instance as represented in SEQ ID NO10), a homologue thereof, or a fragment thereof comprising theRNA-binding domain, said fragment preferably comprising amino acids 1 to56 (SEQ ID NO 12) of the aminoterminal sequence of the LicT protein.

In yet another preferred embodiment the present invention relates to amethod for producing an RNA delivery molecule, wherein the RNA bindingdomain of said delivery molecule comprises a polypeptide comprising thebacteriophage MS2 coat protein (for instance as represented in SEQ ID NO14), a homologue thereof, or a fragment thereof comprising theRNA-binding domain.

In addition, the present invention also relates to methods for producinga chimeric RNAi molecule described herein comprising:

-   -   introducing into a host cell an isolated DNA molecule encoding        any of the chimeric    -   RNAi molecules of the invention;    -   growing the host cell under conditions suitable for expression        of the chimeric RNAi molecule; and

isolating the chimeric RNAi molecule produced by the host cell.

In a preferred embodiment the present invention relates to a method forproducing a chimeric RNAi molecule wherein the chimeric RNAi moleculecomprises:

-   -   at least one nucleotide sequence recognized by an RNA-binding        protein or RNA-binding domain, for instance as represented in        any of SEQ ID NOs 15 to 40, or an RNA secondary structure as        represented in FIG. 41 or 42, and    -   at least one nucleotide sequence corresponding to a target        nucleotide sequence of a target gene of a pest species.

According to another preferred embodiment, the present invention relatesto a method for producing a chimeric RNAi molecule wherein the chimericRNAi molecule has a sequence as represented any of FIGS. 43 to 54 (anyof SEQ ID NOs 41 to 52) or FIGS. 56 to 58 (any of SEQ ID NOs 54 to 56),or any of FIGS. 60 to 74 (any of SEQ ID NOs 57 to 71), or any of FIGS.76 to 90 (any of SEQ ID NOs 72 to 86).

In addition, the present invention also relates to methods for producinga complex comprising an RNA delivery molecule as defined herein, and atleast one chimeric RNAi molecule as defined herein comprising:

-   -   introducing into a host cell an isolated nucleic acid encoding        any of the RNA delivery molecules of the invention and an a        chimeric RNAi molecule as defined herein; provided that the        chimeric RNAi molecule comprises a sequence which is        specifically recognized and able to be bound by the RNA delivery        molecule,    -   growing the host cell under conditions suitable for expression        of the RNA delivery molecule and the chimeric RNAi molecule; and    -   isolating the complex of the RNA delivery molecule with the        chimeric RNAi molecule produced by the host cell.

In the context of preparing an RNA delivery molecule, a chimeric RNAimolecule or a complex comprising an RNA delivery molecule as definedherein, the term “host cell” may any prokaryotic or eukaryotic cell,such as a bacterial, insect, fungal, plant or animal cell. In morespecific embodiment the invention relates to methods for producingspecific complexes as represented in Table 3 or 4.

According to another embodiment, the present invention also relates to akit comprising at least one RNA delivery molecule as described herein.According to yet another embodiment, the present invention also relatesto a kit comprising at least one chimeric RNAi molecule as describedherein. According to yet another embodiment, the present invention alsorelates to a kit comprising a nucleic acid encoding an RNA deliverymolecule as described herein. The invention also relates to a kitcomprising any vector described herein comprising a nucleic acidencoding any of the RNA delivery molecules and/or any of thecorresponding RNAi molecule as described herein. It should be understoodthat these nucleic acid sequences according to the present invention maybe comprised in one or in several separate vectors.

In another preferred embodiment, the invention relates to a kitcomprising a composition or a complex as defined herein. Suitablecombinations of specific RNA delivery molecule with chimeric RNAimolecule in such compositions or complexes are represented in Tables 5and 6.

It is further contemplated that the “composition” of the invention maybe supplied as a “kit-of-parts” comprising the RNA delivery molecule andthe chimeric RNAi molecule in one or in separate containers, and asuitable diluent or carrier for the RNA in a further separate container.The invention also relates to the supply of the RNA delivery moleculeand/or the chimeric RNAi molecule alone without any further components.In these embodiments the RNA delivery molecule and/or the chimeric RNAimolecule may be supplied in a concentrated form, such as a concentratedaqueous solution. It may even be supplied in frozen form or infreeze-dried or lyophilized form. The latter may be more stable for longterm storage and may be de-frosted and/or reconstituted with a suitablediluent immediately prior to use.

The present invention further relates to the medical use of any of thedelivery molecule, chimeric RNAi molecules, constructs, nucleotidesequences, recombinant DNA constructs or compositions thereof describedherein.

In one specific embodiment, the composition is a pharmaceutical orveterinary composition for treating or preventing pest infections ofhumans or animals, respectively. Such compositions will comprise atleast one RNA delivery molecule and at least one chimeric RNAi moleculeaccording to the invention, wherein the chimeric RNAi molecule comprisesdouble-stranded RNA comprising annealed complementary strands, one ofwhich has a nucleotide sequence which is complementary to at least partof a target nucleotide sequence of a target gene of a pest species to bedown-regulated and at least one carrier, excipient suitable forpharmaceutical use or for veterinary use, respectively.

The composition may be a composition suitable for topical use, such asapplication on the skin of an animal or human, for example as liquidcompositions to be applied to the skin as drops, or by brushing, or aspray, also creams, ointments, etc. for topical application andtransdermal patches.

Other conventional pharmaceutical dosage forms may also be produced,including tablets, capsules, pessiaries, suppositories, etc. The chosenform will depend upon the nature of the pest species and hence thenature of the disease it is desired to treat.

Preferred examples of pest species causing infections in human andanimal are fungi. Target human pathogenic and animal pathogenic fungiinclude, but are not limited to the following:

In humans: Candida spp., particularly Candida albicans; Dermatophytes,including Epidermophyton spp., Trichophyton spp., and Microsporum spp.;Aspergillus spp., particularly Aspergillus flavus, Aspergillusfumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreusgroup; Blastomyces dermatitidis; Coccidioides immitis; Crytococcusneoformans; Histoplasma capsulatum Var. capsulatum or Var. duboisii;Sporothrix schenckii; Fusarium spp.; Scopulariopsis brevicaulis;Fonsecaea spp.

In animals: Candida spp.; Microsporum spp., particularly Microsporumcanis, Microsporum gypseum; Trichophyton mentagrophytes; Apergillusspp.; Cryptococcus neoformans.

The composition may be a composition suitable for agronomical use, suchas a spray, a coating, a powder and the like. In particular, the presentinvention provides pesticidal composition developed to be used inagriculture or horticulture. These pesticidal compositions may beprepared in a manner known per se. For example, the active compounds canbe converted into the customary formulations, such as solutions,emulsions, wettable powders, water dispersible granules, suspensions,powders, dusting agents, foaming agents, pastes, soluble powders,granules, suspo-emulsion concentrates, microcapsules, fumigants, naturaland synthetic materials impregnated with active compound and very finecapsules and polymeric substances.

Furthermore, the pesticidal compositions according to the presentinvention may comprise a synergist. The RNA delivery molecule and/orchimeric RNAi molecules, constructs, nucleotide sequences, orcompositions thereof according to the invention, as such or in theirformulations, can also be used in a mixture with known fungicides,bactericides, acaricides, nematicides or insecticides, to widen, forexample, the activity spectrum or to prevent the development ofresistance. In many cases, this results in synergistic effects, i.e. theactivity of the mixture exceeds the activity of the individualcomponents.

Additionally the RNA delivery molecule and/or chimeric RNAi molecules,constructs, nucleotide sequences or compositions thereof according tothe invention, as such or in their formulations or above-mentionedmixtures, can also be used in a mixture with other known activecompounds, such as herbicides, fertilizers and/or growth regulators.

The present invention also relates to fibrous pesticide composition andits use as pesticide, wherein the fibrous composition comprises anon-woven fiber and an effective amount of at least one of the RNAdelivery molecule and/or chimeric RNAi molecules, nucleotide sequences,recombinant DNA constructs or compositions thereof described herein,covalently attached or stably adsorbed to the fiber.

In a further particular embodiment, the fiber is biodegradable and theadsorbed the RNA delivery molecule and/or chimeric RNAi molecules orcompositions thereof as described herein, can be slowly released into alocalized area of the environment to control pests in that area over aperiod of time.

The present invention also encompasses solid formulations ofslow-release pesticidal composition comprising the molecules orconstructs as described herein, and their use as pesticide. Theformulations release the RNA delivery molecules and/or the chimeric RNAimolecule as described herein (a) into the environment (soil, aqueousmedium, plants) in a controlled and slow fashion (complete releasewithin several days up to a few months).

The present invention also relates to surfactant-diatomaceous earthcompositions for pesticidal use in the form of dry spreadable granulescomprising at least one RNA delivery molecule and/or at least onechimeric RNAi molecule as described herein. The granules comprises inaddition to the diatomaceous earth, a surfactant composition designed toprovide binding, rewetting and disintegration properties to thegranules. By diatomaceous earth is meant a silica material characterizedby a large surface area per unit volume. Diatomaceous earth is anaturally occurring material and consists mainly of accumulated shellsor frustules of intricately structured amorphous hydrous silica secretedby diatoms.

The present invention also provides solid, water-insoluble lipospheresand their use as pesticide, wherein said lipospheres are formed of asolid hydrophobic core having a layer of a phospholipid embedded on thesurface of the core, containing at least one RNA delivery moleculeand/or chimeric RNAi molecule as described herein in the core, in thephospholipid, adhered to the phospholipid, or a combination thereof.

The pesticidal compound containing lipospheres have several advantagesincluding stability, low cost of reagents, ease of manufacture, highdispersibility in an aqueous medium, a release rate for the entrappedcompound that is controlled by the phospholipid coating and the carrier.

The invention further relates to pesticidal formulations in the form ofmicrocapsules having a capsule wall made from a urea/dialdehydeprecondensate and comprising at least one RNA delivery molecule and/orchimeric RNAi molecule as described herein.

In another specific embodiment, the composition may be a coating thatcan be applied to a substrate in order to protect said substrate frominfestation by a pest species, such as a fungus and/or to prevent,arrest or reduce growth of the pest species on the substrate and therebyprevent damage caused by said pest species. In this embodiment, thecomposition can be used to protect any substrate or material that issusceptible to infestation by or damage caused by a pest species, forexample foodstuffs and other perishable materials, and substrates suchas wood. Preferred target pest species for this embodiment is are fungusspecies, including, but are not limited to, the following: Stachybotrysspp.; Apergillus spp.; Alternaria spp.; Cladosporium spp.

In this embodiment the composition will comprise at least one RNAdelivery molecule and at least one chimeric RNAi molecule according tothe invention, wherein the chimeric RNAi molecule comprisesdouble-stranded RNA comprising annealed complementary strands, one ofwhich has a nucleotide sequence which is complementary to at least partof a target nucleotide sequence of a target gene of the pest species tobe down-regulated and optionally at least one carrier, excipientsuitable for the intended use.

The nature of the excipients and the physical form of the compositionmay vary depending upon the nature of the substrate that it is desiredto treat. For example, the composition may be a liquid that is brushedor sprayed onto or imprinted into the material or substrate to betreated, or a coating that is applied to the material or substrate to betreated.

The present invention further encompasses a method for treating and/orpreventing fungal infestation on a substrate comprising applying aneffective amount of any of the compositions described herein to saidsubstrate.

The present invention also relates to methods for treating and/orpreventing pest infestation on a substrate comprising applying aneffective amount of a RNA delivery molecule and/or a chimeric RNAimolecule, nucleic acid, vector, or a composition thereof as describedherein to said substrate.

The present invention also relates to methods for treating and/orpreventing pest growth and/or pest infestation of a plant or propagativeor reproductive material of a plant comprising applying an effectiveamount of a RNA delivery molecule and/or a chimeric RNAi molecule,nucleic acid, vector, or a composition thereof as described herein to aplant or to propagation or reproductive material of a plant.

In another embodiment, the invention relates to a method for controllingpest growth on a cell or an organism or for preventing pest infestationof a cell or an organism susceptible to infection to said pest species,comprising contacting said pest species with any of the RNA deliverymolecule and/or the chimeric RNAi molecule, nucleic acid, vector, or acomposition thereof described herein, whereby the dsRNA is taken up bysaid pest species and thereby controls growth or prevents infestation.

Bacteria can be engineered to produce any of the dsRNA or dsRNAconstructs of the invention. These bacteria can be eaten by the pestspecies. When taken up, the dsRNA can initiate an RNAi response, leadingto the degradation of the target mRNA and weakening or killing of thefeeding pest.

Therefore, in a more specific embodiment, said RNA delivery moleculeand/or the chimeric RNAi molecule is expressed by a prokaryotic, such asa bacterial, or eukaryotic, such as a yeast, host cell or host organism.

Some bacteria have a very close interaction with the host plant, such assymbiotic Rhizobium with the Leguminosea (for example Soy). Suchrecombinant bacteria could be mixed with the seeds (ie coating) and usedas soil improvers. Alternatively, dsRNA producing bacteria can besprayed directly onto the crops, for instance Bacillus thuringiensisspecies. Possible applications include intensive greenhouse cultures,for instance crops that are less interesting from a GMO point of view,as well as broader field crops such as soy.

This approach has several advantages, eg: since the problem of possibledicing by a plant host is not present, it allows the delivery of dsRNAinto the gut lumen of the feeding pest; the use of bacteria asinsecticides does not involve the generation of transgenic crops,especially for certain crops where transgenic variants are difficult toobtain; there is a broad and flexible application in that differentcrops can be simultaneously treated on the same field and/or differentpests can be simultaneously targeted, for instance by combiningdifferent bacteria producing distinct dsRNAs.

According to another specific embodiment, the invention encompasses theGMO approaches and thus relates to a method as described above whereinsaid double-stranded RNA is expressed by said cell or organism infestedwith or susceptible to infestation by said pest species, for instancesaid cell is a plant cell or said organism is a plant.

The invention further relates to a method for increasing plant yieldcomprising introducing in a plant any of the RNA delivery moleculeand/or chimeric RNAi molecule, nucleic acid, vector or compositionthereof in an expressible format.

It should be understood that in the above described compositions andkits of the invention comprising both an RNA delivery molecule and achimeric RNAi molecule, the sequences of the molecules should be suchthat the RNA binding protein or RNA-binding domain in the RNA deliverymolecule recognizes and binds to the RNA recognition site, specific forthe RNA binding protein or RNA-binding domain, in the chimeric RNAimolecule.

The invention will be further understood with reference to the followingnon-limiting examples.

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques used in recombinant DNA technology,molecular biology, biological testing, and the like, which are withinthe skill of the art. Such techniques are explained fully in theliterature.

In Example 1, several constructs for RNA delivery molecules aredescribed including possible ways for making them. In Example 2, severalconstructs for chimeric RNAi molecules are described. It should be clearthat the present invention is not limited to the specific constructsexemplified herein.

Example 1 Non-Limiting Examples of RNA Delivery Vehicles According tothe Invention

The RNA delivery vehicle consists of a targeting protein such astransferrin of the target species fused to an RNA binding protein suchas MS2, Nun or licT. The organization of the RNA delivery vehicles is:HIS-RNA-binding protein/domain-linker-transferrin.

Genbank accession numbers:

-   -   Bacteriophage λ Nun protein: P18683, VNBPHK    -   Bacillus subtilis LicT protein: P39805, S47216, BAA11696,        CAA82194    -   Bacteriophage MS2 coat protein: P03612, CAA23989, NP_(—)040648,        VCBPM2, 721932A    -   Human transferrin protein: P02787, 043890 Q9NQB8 Q9UHV0    -   Drosophila melanogaster transferrin homolog Tsf1: NM_(—)078677    -   Drosophila melanogaster transferrin homolog Tsf2: NM_(—)079320    -   Galanthus nivalis agglutinin: P30617, JE0136, S19735    -   Potato Leafroll virus capsid protein: VCVQL2, AAL77965, AAL77957    -   Potato Leafroll virus read-through protein: NP_(—)056751,        AAN38829, AAL77959

1.1. Full-Length Nun Protein Linked to Transferrin

Step 1: To obtain the Nun gene sequence, PCR is performed with primerspR11 and pR12 on bacteriophage λ DNA and the PCR product is cloned intothe TOPO TA Cloning® vector (Invitrogen) and sequence verified.

-   -   pR11 is a sense primer starting at the ATG startcodon of the Nun        gene; pR12 is an antisense primer starting at the TAA stopcodon        of the Nun gene.        Step 2: For cloning the Nun ORF behind the His-tag, PCR is        performed with primers pR13 and pR14 on the vector resulting        from step 1. The PCR product is digested with NcoI and SpeI,        cloned into the pFastBac™ HTa vector (Invitrogen) digested with        NcoI and SpeI, and sequence verified.    -   pR13 is the same primer as pR11 but the ATG startcodon of the        Nun gene is preceded by a NcoI restriction site; pR14 is the        same primer as pR12 but lacks the TAA stopcodon of the Nun gene;        this gene-specific sequence is preceded by the linker encoding        sequence and a SpeI site.        Step 3: To obtain the transferrin sequence, PCR is performed        with primers pE11 and pE12 on a human cDNA library and the PCR        product is cloned into the TOPO TA Cloning® vector and sequence        verified.    -   pE11 is a sense primer starting at the ATG startcodon of the        transferrin gene; pE12 is an antisense primer starting at the        TAA stopcodon of the transferrin gene.        Step 4: To make the Nun-transferrin fusion gene, PCR is        performed with primers pE13 and pE14 on the vector resulting        from step 3. The PCR product is digested with SpeI and XbaI,        cloned into the vector resulting from step 2 digested with SpeI        and XbaI, and sequence verified.    -   pE13 is the same primer as pE11 but the ATG startcodon of the        transferrin gene is preceded by a SpeI restriction site; pE14 is        the same primer as pE12 but the TAA stopcodon of the transferrin        gene is preceded by an XbaI restriction site.

1.2. N-Terminal 47 Amino Acids of Nun Protein Linked to Transferrin

To obtain this fusion construct the same cloning steps are performed asin Example 1.1, but in Step 2 PCR primer pR14 is replaced by PCR primerpR18.

-   -   pPR18 is an antisense primer starting at codon 47 (nucleotides        139-141) instead of at the TAA stopcodon; again, this        gene-specific sequence is preceded by the linker encoding        sequence and a SpeI site.

1.3. Amino Acids 13-47 of Nun Protein Linked to Transferrin

To obtain this fusion construct the same cloning steps are performed asin Example 1.1, but in Step 2 PCR primer pR13 is replaced by PCR primerpR15 and pR14 is replaced by PCR primer pR18.

-   -   pR15 is a sense primer starting at nucleotide 37 (codon 13) of        the Nun gene; this gene-specific sequence is preceded by a NcoI        restriction site; pR18 is the same as in Example 1.2.

1.4. Amino Acids 22-47 of Nun Protein Linked to Transferrin

To obtain this fusion construct the same cloning steps are performed asin Example 1.1, but in Step 2 PCR primer pR13 is replaced by PCR primerpR17 and pR14 is replaced by PCR primer pR18.

-   -   pR17 is a sense primer starting at nucleotide 64 (codon 22) of        the Nun gene; this gene-specific sequence is preceded by a NcoI        restriction site; pR18 is the same as in Example 1.2.        1.5. Drosophila melanogaster transferrin Drosophila melanogaster        contains two transferrin homologs, Tsf1 and Tsf2. Both        Drosophila melanogaster transferrin homologs are fused to the        full-length Nun protein or to either of the Nun dsRNA-binding        domain fragments following the same strategies as in Examples        2.1 through 2.4, but replacing the human transferrin-specific        sequence of the primers pE11 through pE14 with Drosophila        melanogaster transferrin-specific sequence.

1.6. Full-Length LicT Protein Linked to Transferrin

Step 1: To obtain the LicT gene sequence, PCR is performed with primerspR21 and pR22 on Bacillus subtilis DNA and the PCR product is clonedinto the TOPO TA Cloning® vector and sequence verified.

-   -   pR21 is a sense primer starting at the ATG startcodon of the        LicT gene; pR22 is an antisense primer starting at the TAA        stopcodon of the LicT gene.        Step 2: For cloning the LicT ORF behind the His-tag, PCR is        performed with primers pR23 and pR24 on the vector resulting        from step 1. The PCR product is digested with BspHI and SpeI,        cloned into the pFastBac™ HTa vector digested with NcoI and        SpeI, and sequence verified.    -   pR23 is the same primer as pR21 but the ATG startcodon of the        LicT gene is preceded by a BspHI restriction site; pR24 is the        same primer as pR22 but lacks the TAA stopcodon of the LicT        gene; this gene-specific sequence is preceded by the linker        encoding sequence and a SpeI site.        Step 3 and Step 4 are identical to Step 3 and Step 4 of Example        1.1.

1.7. N-Terminal 56 Amino Acids of LicT Protein Linked to Transferrin

To obtain this fusion construct the same cloning steps are performed asin Example 1.6, but in Step 2 PCR primer pR24 is replaced by PCR primerpR26.

-   -   pR26 is an antisense primer starting at codon 56 (nucleotides        166-168) instead of at the TAA stopcodon; again, this        gene-specific sequence is preceded by the linker encoding        sequence and a SpeI site.        1.8. Drosophila melanogaster Transferrin

Drosophila melanogaster contains two transferrin homologs, Tsf1 andTsf2. Both Drosophila melanogaster transferrin homologs are fused to thefull-length LicT protein or to its N-terminal dsRNA-binding domainfollowing the same strategies as in Examples 1.6 and 1.7, but replacingthe human transferrin-specific sequence of the primers pE11 through pE14with Drosophila melanogaster transferrin-specific sequence.

1.9 Full-Length MS2 Protein Linked to Human Transferrin

Step 1: To obtain the MS2 gene sequence, PCR is performed with primerspR31 and pR32 on bacteriophage MS2 DNA and the PCR product is clonedinto the TOPO TA Cloning® vector and sequence verified.

-   -   pR31 is a sense primer starting at the ATG startcodon of the MS2        coat protein gene; pR32 is an antisense primer starting at the        TAA stopcodon of the MS2 coat protein gene.        Step 2: For cloning the MS2 coat protein ORF behind the His-tag,        PCR is performed with primers pR33 and pR34 on the vector        resulting from step 1. The PCR product is digested with NcoI and        SpeI, cloned into the pFastBac™ HTa vector digested with NcoI        and SpeI, and sequence verified.    -   pR33 is the same primer as pR31 but the ATG startcodon of the        MS2 coat protein gene is made part of a NcoI restriction site;        pR34 is the same primer as pR32 but lacks the TAA stopcodon of        the MS2 coat protein gene; this gene-specific sequence is        preceded by the linker encoding sequence and a SpeI site.        Step 3 and Step 4 are identical to Step 3 and Step 4 of Example        1.1.        1.10. Drosophila melanogaster Transferrin

Drosophila melanogaster contains two transferrin homologs, Tsf1 andTsf2. Both Drosophila melanogaster transferrin homologs are fused to theMS2 coat protein protein following the same strategies as in Example1.9, but replacing the human transferrin-specific sequence of theprimers pE11 through pE14 with Drosophila melanogastertransferrin-specific sequence.

1.11. Full-Length Nun Protein Linked to Galanthus nivalis Agglutinin

Step 1 and Step 2 are identical to Step 1 and Step 2 of Example 1.1.

Step 3: To obtain the Galanthus nivalis agglutinin sequence, PCR isperformed with primers pE21 and pE22 on a Galanthus nivalis (CommonSnowdrop) cDNA library and the PCR product is cloned into the TOPO TACloning® vector and sequence verified.

-   -   pE21 is a sense primer starting at the ATG startcodon of the        Galanthus nivalis agglutinin gene; pE22 is an antisense primer        starting at the TAA stopcodon of the Galanthus nivalis        agglutinin gene.        Step 4: To make the Nun—Galanthus nivalis agglutinin fusion        gene, PCR is performed with primers pE23 and pE24 on the vector        resulting from step 3. The PCR product is digested with SpeI and        XbaI, cloned into the vector resulting from step 2 digested with        SpeI and XbaI, and sequence verified.    -   pE23 is the same primer as pE21 but the ATG startcodon of the        Galanthus nivalis agglutinin gene is preceded by a SpeI        restriction site; pE24 is the same primer as pE22 but the TAA        stopcodon of the Galanthus nivalis agglutinin gene is preceded        by an XbaI restriction site.        1.12. N-Terminal 47 Amino Acids of Nun Protein Linked to        Galanthus nivalis Agglutinin

To obtain this fusion construct the same cloning steps are performed asin Example 1.11, but in Step 2 PCR primer pR14 is replaced by PCR primerpR18.

-   -   pR18 is the same as in Example 1.2.        1.13. Amino Acids 13-47 of Nun Protein Linked to Galanthus        nivalis Agglutinin

To obtain this fusion construct the same cloning steps are performed asin Example 1.11, but in Step 2 PCR primer pR13 is replaced by PCR primerpR15 and pR14 is replaced by PCR primer pR18.

-   -   pR15 is the same as in Example 1.3.; pR18 is the same as in        Example 1.2.

1.14. Amino Acids 22-47 of Nun Protein Linked to Galanthus NivalisAgglutinin

To obtain this fusion construct the same cloning steps are performed asin Example 1.11, but in Step 2 PCR primer pR13 is replaced by PCR primerpR17 and pR14 is replaced by PCR primer pR18.

-   -   pR17 is the same as in Example 1.4.; pR18 is the same as in        Example 1.2.        1.15. Full-Length LicT Protein Linked to Galanthus nivalis        Agglutinin        Step 1 and Step 2 are identical to Step 1 and Step 2 of Example        1.6.        Step 3 and Step 4 are identical to Step 3 and Step 4 of Example        1.11.

1.16. N-Terminal 56 Amino Acids of LicT Protein Linked to GalanthusNivalis Agglutinin

To obtain this fusion construct the same cloning steps are performed asin Example 1.15, but in Step 2 PCR primer pR24 is replaced by PCR primerpR26.

-   -   pR26 is the same as in Example 1.7.

1.17 Full-Length MS2 Protein Linked to Human Galanthus NivalisAgglutinin

Step 1 and Step 2 are identical to Step 1 and Step 2 of Example 1.9.Step 3 and Step 4 are identical to Step 3 and Step 4 of Example 1.11.

1.18. Full-Length Nun Protein Linked to Potato Leafroll Virus CapsidProtein

Step 1 and Step 2 are identical to Step 1 and Step 2 of Example 1.1.Step 3: To obtain the Potato Leafroll virus capsid protein genomicsequence, PCR is performed with primers pE31 and pE32 on Potato Leafrollvirus cDNA and the PCR product is cloned into the TOPO TA Cloning®vector and sequence verified.

-   -   pE31 is a sense primer starting at the ATG startcodon of the        Potato Leafroll virus capsid protein gene; pE32 is an antisense        primer starting at the TAA stopcodon of Potato Leafroll virus        capsid protein gene.        Step 4: To make the Nun—Potato Leafroll virus capsid protein        fusion gene, PCR is performed with primers pE33 and pE34 on the        vector resulting from step 3. The PCR product is digested with        SpeI and XbaI, cloned into the vector resulting from step 2        digested with SpeI and XbaI, and sequence verified.    -   pE33 is the same primer as pE31 but the ATG startcodon of the        Potato Leafroll virus capsid protein gene is preceded by a SpeI        restriction site; pE34 is the same primer as pE32 but the TAA        stopcodon of the Potato Leafroll virus capsid protein gene is        preceded by an XbaI restriction site.

1.19. N-Terminal 47 Amino Acids of Nun Protein Linked to Potato LeafrollVirus Capsid Protein

To obtain this fusion construct the same cloning steps are performed asin Example 1.18, but in Step 2 PCR primer pR14 is replaced by PCR primerpR18.

-   -   pR18 is the same as in Example 1.2.

1.20. Amino Acids 13-47 Amino Acids of Nun Protein Linked to PotatoLeafroll Virus Capsid Protein

To obtain this fusion construct the same cloning steps are performed asin Example 1.18, but in Step 2 PCR primer pR13 is replaced by PCR primerpR15 and pR14 is replaced by PCR primer pR18.

1.21. Amino Acids 22-47 Amino Acids of Nun Protein Linked to PotatoLeafroll Virus Capsid Protein

To obtain this fusion construct the same cloning steps are performed asin Example 1.18, but in Step 2 PCR primer pR13 is replaced by PCR primerpR17 and pR14 is replaced by PCR primer pR18.

-   -   pR15 is the same as in Example 1.3.;pR18 is the same as in        Example 1.2.

1.22 Potato Leafroll Virus Read-Through Protein

Potato Leafroll virus read-through protein is fused to the full-lengthNun protein or to either of the Nun dsRNA-binding domain fragmentsfollowing the same strategies as in Examples 1.18 through 1.21, but inStep 3 PCR primer pE32 is replaced by PCR primer pE36 and in Step 4 PCRprimer pE34 is replaced by PCR primer pE38.

-   -   pE36 is an antisense primer starting at the TAA stopcodon of        Potato Leafroll virus read-through protein gene; pE38 is the        same primer as pE36 but the TAA stopcodon of the Potato Leafroll        virus read-through protein gene is preceded by an XbaI        restriction site.

1.23. Full-Length LicT Protein Linked to Potato Leafroll Virus CapsidProtein

Step 1 and Step 2 are identical to Step 1 and Step 2 of Example 1.6.Step 3 and Step 4 are identical to Step 3 and Step 4 of Example 1.18.

1.24. N-Terminal 56 Amino Acids of LicT Protein Linked to PotatoLeafroll Virus Capsid Protein

To obtain this fusion construct the same cloning steps are performed asin Example 1.23, but in Step 2 PCR primer pR24 is replaced by PCR primerpR26.

-   -   pR26 is the same as in Example 1.7.

1.25 Potato Leafroll Virus Read-Through Protein

Potato Leafroll virus read-through protein is fused to the full-lengthLicT protein or to its N-terminal dsRNA-binding domain following thesame strategies as in Examples 1.23 and 1.24, but in Step 3 PCR primerpE32 is replaced by PCR primer pE36 and in Step 4 PCR primer pE34 isreplaced by PCR primer pE38.

-   -   pR36 and pR38 are the same as in Example 1.22.

1.26 Full-Length MS2 Protein Linked to Potato Leafroll Virus CapsidProtein

Step 1 and Step 2 are identical to Step 1 and Step 2 of Example 1.9.Step 3 and Step 4 are identical to Step 3 and Step 4 of Example 1.18.

1.27 Full-Length MS2 Protein Linked to Potato Leafroll VirusRead-Through Protein

Potato Leafroll virus read-through protein is fused to the full-lengthMS2 protein following the same strategies as in Example 2.26, but inStep 3 PCR primer pE32 is replaced by PCR primer pE36 and in Step 4 PCRprimer pE34 is replaced by PCR primer pE38.

-   -   pR36 and pR38 are the same as in Example 1.22.

Example 2 Non-Limiting Examples for the Construction of Chimeric RNAiMolecules that Recognize RNA Delivery Molecules According to theInvention

The chimeric RNAi molecule is principally a hairpin construct thatcontains at least one target RNA sequence and, in the loop(s) or at the3′ or 5′ end, at least one recognition sequence for an RNA-bindingprotein, such as for instance MS2, Nun or licT.

Target genes from the target species are cloned accordingly. Examples oftarget genes in C. elegans include 6-tubulin (Genbank accessionNM_(—)066966), sup-35 (Genbank accession number X73845) unc-22 (Genbankaccession Z73899) and 60S ribosomal protein L9 (Genbank accessionNM_(—)066259), see Table 7. The length of the tested target RNA sequenceis 80-450 base pairs.

TABLE 7 Overview of the examples: different genes in four differentplant pest species, Caenorhabditis elegans, Nilaparvata lugens,Diabrotica virgifera, Meloidogyne incognita, and Magnaporthe grisea.Nilaparvata lugens C. elegans M. incognita Diabrotica virgifera M.grisae Target gene β-tubulin β-tubulin β-tubulin β-tubulin rpl-9,sup-35, rpl-9 rpl-9 rpl-9 unc-22 In vitro uptake Drinking assay Drinkingassay Bio Assay Soaking In planta X Hairy root Whole plant Hairy root,assay, whole whole plant plant

Promoters useful for the expression of dsRNA are a promoter from an RNAPoll, an RNA PollI, an RNA PollII, T7 RNA polymerase or SP6 RNApolymerase.

Several non-limiting constructs of chimeric RNAi molecules according tothe invention are exemplified in FIGS. 43 to 54, FIGS. 56 to 58, FIGS.60 to 74 and FIGS. 76 to 90. The chimeric RNAi molecules consist of aleast two modules: at least one dsRNA sequence against the pest targetgene, and (at least one) sequence recognized by the Lict, Nun or MS2protein or RNA binding domain thereof. The target sequence can be anyspecies-specific sequence that induces lethality or any other severephenotype in the target species. The RNA binding protein recognitionsequence is recognized by the RNA binding protein, such as LicT, Nun orMS2. In case of two recognition sequences in the molecule, these can berecognized by the same RNA binding protein, or by two different RNAbinding proteins. In the examples of FIGS. 43 to 54, 60 to 71 and 76 to87, each of these constructs comprises at least one recognition site foran RNA-binding domain and a species-specific target sequence fordown-regulation of tubulin (see FIG. 55, 59 or 75). In the examples ofFIGS. 56 to 58, 72 to 74 and 88 to 90 each of these constructs comprisesat least one recognition site for an RNA-binding domain and aspecies-specific target sequence for down-regulation of unc-22 (see FIG.55).

Example 3 Non-Limiting Examples of C. elegans Assays to Measure theEfficiency of dsRNA Delivery to Target Cells Using the ConstructsAccording to the Invention

In the following example, several assays are described which are used tofollow efficacy of RNAi in pest species.

A) Preparation of RNA Vehicle Complex (RVC)

The RNA vehicle complex principally consists of an RNAi mediatingmolecule (dsRNA or hairpin as described in example 2) to induce RNAi inthe target organism and an RNA delivery vehicle (as described inexample 1) to increase the efficiency of endo- and/or transcytosis.

RNA Delivery Vehicle Production and Purification of the RNA DeliveryVehicle in the Baculovirus Expression System:

RNA delivery vehicles, as described in Example 1, are produced in insectSf9 cells after transfection with recombinant baculoviruses using theBac-to-Bac® Baculovirus Expression System (Invitrogen). The His-taggedRNA delivery vehicles are protein purified over a HiTrap™ Chelating HPnickel column (Amersham Boisciences). The His-tag is removed by cleavagewith recombinant TEV protease (Invitrogen). Protein purification withnickel-chelating resin and rTEV cleavage are also used when RNA deliveryvehicle protein is obtained after expression in bacteria or the yeastPichia Pastoris.

RNAi Mediating Molecule Expression and Purification Using BacteriaExpression System

The RNAi mediating molecule is obtained using T7 polymerasetranscription of the template DNA following the provided protocol of RNAdsRNA kit manufacturers (e.g. T7 RiboMAX™ Express RNAi System, Promegaor obtained by Megascript RNAi™ kit, Ambion)

B) Administration of RNA Vehicle Complex (RVC) to C. elegans

The RVC complex can be delivered to the C. elegans gut by feeding onagar plates, soaking in liquid medium or injection into the gut lumen.

RVC Delivery into C. elegans by Soaking and Injection

RNAi is induced by injection of dsRNA into C. elegans or by feeding C.elegans a solution with dsRNA, called soaking (Gonczy P, Echeverri C,Oegema K, Coulson A, Jones S J, Copley R R, Duperon J, Oegema J, BrehmM, Cassin E, Hannak E, Kirkham M, Pichler S, Flohrs K, Goessen A, LeidelS, Alleaume A M, Martin C, Ozlu N, Bork P, Hyman A A. Nature. 2000 408page 331-6; Maeda I, Kohara Y, Yamamoto M, Sugimoto A. Curr Biol. 200111 page 171-6). These methods can also be used to induce RNAi by feedingwith RVC. The RVC complex is prepared by mixing the RNA vehicle proteinand the RNA construct in physiological buffer. This solution is eitherinjected into the gut lumen of C. elegans or C. elegans is soaked in asolution containing the RVC complex In the latter case, the C. eleganswould drink the RVC in the solution and consequently ingest it into thegut. A variation of this theme is to add the RVC solution on top of E.coli seeded agar plates on which C. elegans can feed.

RVC Delivery by Feeding

RNAi by feeding is induced in C. elegans by feeding with E. coli, whichexpresses dsRNA against the target gene (Fraser A G, Kamath R S,Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Nature. 2000 408page 325-30). Similarly, E. coli is co-transformed with a vectorexpressing the chimeric RNAi molecule against the target gene and avector expressing the RNA delivery vehicle. C. elegans feeds on these E.coli and ingests RNA and protein to form the RVC. Upon digestion of E.coli, the RVC is released into the gut lumen.

All three delivery methods provide the RVC complex to the apical gutsurface. The vehicle binds to apical surface proteins that mediatereceptor-mediated endocytosis or are endocytosed specifically. Thisbinding greatly enhances absorption into the gut.

C) Measurement of Improved RNAi Effectiveness in C. elegans

The effectiveness of the RVC to induce RNAi is measured using a specificand visible movement phenotype, “twitcher”. Down-regulation by RNAi ormutations in the C. elegans gene unc-22, which encodes Twitchin, causesconvulsive muscle contractions called “twichter” phenotype (Benian G Met al. 1989. Sequence of an unusually large protein implicated inregulation of myosin activity in C. elegans. Nature 342: 45-50). In thisexperiment the following test reagents are used:

-   -   1) Standard dsRNA (RNA without vehicle) against unc-22    -   2) RVC consisting of e.g., transferrin (see Example 1, 1.1 and        1.9) and RNA against unc-22 (see Example 2, 2.1)        RVC and standard dsRNA are administered to C. elegans for        example by soaking (as described under A). Different        concentrations of RVC and standard dsRNA are tested in 24 or        96-well plates. Ten to fifty L1 larvae are placed into each well        and grown for several days until adulthood at 25° C., whereby        sufficient food (E. coli) is supplied. Adults are examined for        the presence of the “twitcher” phenotype using a dissection        microscope. Depending on the efficiency of RNAi against unc-22 a        certain fraction of animals of a population will exhibit the        “twitcher” phenotype. The fraction of animals exhibiting the        twitcher phenotype increases with increasing concentration of        dsRNA in a dose-dependent manner. If the RVC complex increases        delivery of effective RNAi mediating molecules by enhanced        endo/transcytosis, the fraction of animals with “twitcher”        phenotype is greater at a given concentration (concentration of        the RNA molecules is identical) compared to standard dsRNA. As a        result, the dose-response curve obtained with RVC is shifted to        lower concentrations. The extent of the shift is a means to        measure changed efficiency of the RVC. Similarly, the same        protocol can be used to test essential genes such as β-tubulin,        which cause growth delay and death.

Example 4 Non-Limiting Examples of an Insect Bio Assay to Measure theEfficiency of dsRNA Delivery to Target Cells Using the ConstructsAccording to the Invention

The effectiveness of the RVC(RNA vehicle complex) to induce RNAi in thetarget pest such as corn root worm or planthopper is measured by itspotential to increase insect mortality. The selected pest gene is anessential gene and it has been demonstrated that knock down by RNAicauses impaired viability or lethality. In this experiment the followingtest reagents are used:

-   -   1) Standard dsRNA (RNA without vehicle) against pest gene    -   2) RVC consisting of e.g., transferrin (see Example 1, 1.5 and        1.10) and RNA (see example 2, 2.x) against pest gene.        RVC and standard dsRNA are applied to the surface of casein and        wheat germ modified artificial diet for corn rootworm        (Marrone, P. G., F. D. Ferri, T. R. Mosley, and L. J.        Meinke. 1985. J. Econ. Entomol. 78:290-293). Different        concentrations of RVC and standard dsRNA are tested in 48 or        96-well plates. Each well is infested with one to six neonate        corn root worms (respectively 50 to 100 animals per condition)        and grown for 4 to 12 days at 25° C. and ˜50% relative humidity.        Similar for planthopper: Infestation with third-instar nymphs or        adults in 24 or 48-well plates, which are grown at 25° C. and        60% relative humidity under long-day (16L/8D) conditions (Ito M,        Okui H, Nakagawa H, Mio S, Kinoshita A, Obayashi T, Miura T,        Nagai J, Yokoi S, Ichinose R, Tanaka K, Kodama S, Iwasaki T,        Miyake T, Takashio M, Iwabuchi J. (2002). Biosci Biotechnol        Biochem. 2002. 66:2406-14). Plates are examined for larval and        adult mortality using a dissection microscope. Increasing        concentration of dsRNA or RVC increases mortality in a        dose-dependent manner. If the RVC complex increases the number        of RNAi mediating molecules by enhanced endo/transcytosis, the        mortality is greater compared to standard dsRNA at a given        concentration. As a result, the dose-response curve obtained        with RVC is shifted to lower concentrations respectively the        LD₅₀ is reduced (LD₅₀ is defined as the concentration that        caused 50% mortality of the test population).

Example 5 Non-Limiting Examples Assay to Monitor dsRNA Delivery toTarget Cells Using Labeled Constructs According to the Invention

Endo/transcystosis is monitored using labeled RNA and labeled vehicles.In this experiment the following test reagents are used:

-   -   A Labeled vehicle    -   B Labeled RNAi    -   C Co-labeled RVC        Reagentsare applied to C. elegans animals and the uptake from        gut lumen (apical) into the gut cell, across the gut cell into        the pseudocoelom (basolateral) is monitored. Fluorecence        technology might apply using FITC labeled vehicel and Cy3        labeled-Uracil incorporated into the RNA molecule. Given the        potential processing during endo/transcytosis and the potential        dilution of RNA molecules in the liquid of the pseudocoelom,        radioactive labeling using for example S³⁵ is preferred.

Results.

The vehicle may accumulate on the apical surface, in the gut cell andpotentially at the basolateral surface (in case the vehicle wastranscytosed). Similarly, labeled RNA may be visible on the gut cellsurface/in the gut cells. It is expected that the RNA delivered via theRVC complex is accumulated significantly higher on/in the gut than pleinRNA proving the concept of improved endo/transcytosis via RVC.

Stability of the dsRNa Constructs of the Present Invention in PlantCells

The stability of the expressed constructs are analyzed with quantitativereal-time PCR to determine the quantity of the expressed construct ofthe invention present in the transgenic plant cell relative to thequantities present in control transgenic plants, expressing a controldsRNA construct. The method to monitor PCR in real-time is describedpreviously and is based on Taqman probes or intercalating dyes (SYBRgreen).

The expressed dsRNA constructs are quantified relative towards astandard dilution series of the template. The results are normalized byusing the quantitative PCR data of a set of housekeeping genes from thesame samples (Vandesompele et al., Genome Biology 2002,3:research0034.1-0034.11).

Example 6 Non-Limiting Examples of an Insect in Planta Assay to Measurethe Efficiency of dsRNA Delivery to Target Cells Using the ConstructsAccording to the Invention

Plant transformation always requires co-transformation of the plant witha construct containing the RNA delivery vehicle and a constructcontaining the RNA mediating molecule. The constructs of the presentinvention are cloned behind the CaMV35S promoter, a root or leavespecific promoter or a feeding site specific promoter in a binary vectorsuitable for plant transformation. The binary vectors are transferred toAgrobacterium rhizogenes by three-parental mating (e.g. by E. coli HB101containing pRK2013 helper plasmid). The binary vectors are transferredfrom E. coli into Agrobacterium tumifaciens. Subsequently crops plants(such as tomato, soybean, cotton or Tobacco) are co-transformed with theconstructs (one containing the RNA delivery vehicle and one containingthe RNA mediating molecule) via Agrobacterium-mediated transformation.As a control, Agrobacterium without binary vector is used. (Example of aprotocol for corn transformation: Frame B R, Shou H, Chikwamba R K,Zhang Z, Xiang C, Fonger T M, Pegg S E, Li B, Nettleton D S, Pei D, WangK. (2002) Agrobacterium tumefaciens-mediated transformation of maizeembryos using a standard binary vector system. Plant Physiol. 129 page13-22.). (Example of a protocol for rice transformation: Breitler J C,Meynard D, Van Boxtel J, Royer M, Bonnot F, Cambillau L, Guiderdoni E.(2004). A novel two T-DNA binary vector allows efficient generation ofmarker-free transgenic plants in three elite cultivars of rice (Oryzasativa L.). Transgenic Res 13:271-87).

A) Corn Rootworm

Plant tissues are transformed with the constructs of the presentinvention and then regenerated into whole plants. Whole-transgenicplants are infested with corn rootworm eggs. Three parameters arescored: (1) Insect mortality, by which after various time points insectsare collected and examined for stage of development and mortalitycompared to non-transformed plants and insecticide treated plants. (2)Plant-resistance, by which the quality of the roots are examined with arating system such as nodes destroyed, roots pruned or undamaged roots.(3) Plant health, by which plant growth and root density of transformedversus non-transformed versus nematicide treated plants are compared. (Aprotocol describing the steps from shot gun mediated corn transformationto corn root worm resistance measurement can be found in Moellenbeck DJ, Peters M L, Bing J W, Rouse J R, Higgins L S, Sims L, Nevshemal T,Marshall L, Ellis R T, Bystrak P G, Lang B A, Stewart J L, Kouba K,Sondag V, Gustafson V, Nour K, Xu D, Swenson J, Zhang J, Czapla T,Schwab G, Jayne S, Stockhoff B A, Narva K, Schnepf H E, Stelman S J,Poutre C, Koziel M, Duck N. (2001). Insecticidal proteins from Bacillusthuringiensis protect corn from corn rootworms. Nat. Biotechnol. 19 page668-72.)

B) Planthopper

Plants (or plant) tissues are transformed with the constructs of thepresent invention and then regenerated into whole plants.Whole-transgenic plants are infested with plant hopper neonates, thirdinstar nymphs or adults. Three parameters are scored: (1) Insectmortality, by which after various time points insects are collected andexamined for stage of development and mortality compared tonon-transformed plants and insecticide treated plants. (2)Plant-resistance, by which the quality of the rice stem and rice leavesare examined with a rating system such as rice stem/leaves have severefeeding damage, rice stem/leaves have limited feeding damage orundamaged steem/leaves. (3) Plant health, by which plant growth, steemlength and leaf quality of transformed versus non-transformed versusnematicide treated plants are compared (a protocol describing the stepsfrom rice transformation to plant hopper bio assay can be found in(Nayak P, Basu D, Das S, Basu A, Ghosh D, Ramakrishnan N A, Ghosh M, SenS K. (1997). Proc Natl Acad Sci USA. 94:2111-6).

1. An RNA delivery molecule consisting of a polypeptide sequencecomprising i) at least one RNA-binding domain, ii) at least onetargeting polypeptide able to bind to a cellular endocytosis and/ortranscytosis receptor molecule, and iii) optionally at least one peptidelinker for linking the RNA binding domain (i) to the targetingpolypeptide (ii).
 2. An RNA delivery molecule according to claim 1,wherein said at least one RNA-binding domain (i) is chosen from: apolypeptide comprising the coliphage HK022 Nun protein (for instance asrepresented in SEQ ID NO 2), a homologue thereof, or a fragment thereofcomprising the RNA-binding domain, said fragment preferably comprisingamino acids 1 to 47 (SEQ ID NO 4) of the aminoterminal sequence, morepreferably comprising amino acids 13 to 47 (SEQ ID NO 6) of theaminoterminal sequence, most preferably comprising amino acids 22 to 47(SEQ ID NO 8) of the aminoterminal sequence, a polypeptide comprisingthe Bacillus subtilis LicT protein (for instance as represented in SEQID NO 10), a homologue thereof, or a fragment thereof comprising theRNA-binding domain, said fragment preferably comprising amino acids 1 to56 (SEQ ID NO 12) of the aminoterminal sequence, and a polypeptidecomprising the bacteriophage MS2 coat protein (for instance asrepresented in SEQ ID NO 14), a homologue thereof, or a fragment thereofcomprising the RNA-binding domain.
 3. An RNA delivery molecule accordingto claim 1, wherein said at least one targeting polypeptide (ii) bindsto a gut cell endocytosis or transcytosis receptor molecule.
 4. An RNAdelivery molecule according to claim 1, wherein said at least onetargeting polypeptide (ii) binds to a tissue cell endocytosis ortranscytosis receptor molecule.
 5. An RNA delivery molecule according toclaim 1, further comprising a purification tag.
 6. A method fordelivering a double-stranded RNA molecule to a pest species, comprisingco-expressing in a plant cell of: a) an RNA delivery molecule accordingto claim 1, and b) an RNA molecule which comprises double-stranded RNAcomprising annealed complementary strands, one of which has a nucleotidesequence which is complementary to at least part of a target nucleotidesequence of a target gene of the pest species, and which furthercomprises a nucleotide sequence which binds to the RNA-binding domain ofthe RNA delivery molecule of (a). feeding said plant cell to said pestspecies.
 7. A method for delivering double-stranded RNA to a pestspecies, comprising feeding the pest species with a mixture of an RNAmolecule and an RNA delivery molecule according to claim 1, wherein theRNA molecule comprises double-stranded RNA comprising annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to at least part of a target nucleotide sequence of atarget gene of the pest species, and which further comprises anucleotide sequence which binds to the RNA-binding domain of the RNAdelivery molecule, whereby the mixture is taken up into the gut of thepest species, and whereby the complex of the double-stranded RNA boundto the RNA delivery molecule is transcytosed and/or endocytosed by a gutcell and/or a tissue cell.
 8. A method for down-regulating expression ofa target gene in a pest species, comprising feeding the pest specieswith a mixture of an RNA molecule and an RNA delivery molecule accordingto claim 1, wherein the RNA molecule comprises double-stranded RNAcomprising annealed complementary strands, one of which has a nucleotidesequence which is complementary to at least part of a target nucleotidesequence of the target gene to be down-regulated, and further comprisesa nucleotide sequence which binds to the RNA-binding domain of the RNAdelivery molecule, whereby the mixture is taken up into the gut of thepest species, and whereby the complex of the double-stranded RNA boundto the RNA delivery molecule is transcytosed and/or endocytosed by a gutcell and/or a tissue cell and thereby down-regulates expression of thetarget gene in a pest cell.
 9. A method for down-regulating expressionof a target gene in a pest species, comprising: co-expressing in a plantcell of: a) an RNA delivery molecule according to claim 1, b) an RNAmolecule which comprises double-stranded RNA comprising annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to at least part of a target nucleotide sequence of thetarget gene to be down-regulated, and which further comprises anucleotide sequence which binds to the RNA-binding domain of the RNAdelivery molecule of (a), and feeding said plant cell to said pestspecies.
 10. A method for producing a transgenic plant that is resistantto a pest species, comprising: co-expressing in a plant cell of: a) anRNA delivery molecule according to claim 1, and b) an RNA molecule whichcomprises double-stranded RNA comprising annealed complementary strands,one of which has a nucleotide sequence which is complementary to atleast part of a target nucleotide sequence of the target gene of saidpest species, and which further comprises a nucleotide sequence whichbinds to the RNA-binding domain of the RNA delivery molecule of (a), andregenerating a plant from said plant cell.
 11. A method according toclaim 6, wherein in (a) the RNA-binding domain comprises the coliphageHK022 Nun protein or a fragment thereof, said fragment comprising theRNA-binding domain of the HK022 Nun protein, and wherein the doublestranded RNA molecule (b) comprises a nucleotide sequence correspondingto the binding motif for binding to the coliphage HK022 Nun protein or afragment thereof.
 12. A method according to claim 11, wherein in (a) theRNA-binding domain is a polypeptide comprising the sequence representedin any of SEQ ID NOs 2, 4, 6 or 8, preferably comprising the sequencerepresented in SEQ ID NO 8, and wherein (b) comprises the binding motifrepresented in SEQ ID NO 15 or
 16. 13. A method according to claim 6,wherein in (a) the RNA binding domain is the Bacillus subtilis LicTprotein or a fragment thereof, said fragment comprising the RNA bindingdomain and wherein the double stranded RNA molecule (b) comprises anucleotide sequence corresponding to the binding motif for binding tothe Bacillus subtilis LicT protein or a fragment thereof.
 14. A methodaccording to claim 13, wherein in (a) the RNA binding domain is apolypeptide comprising the sequence represented in SEQ ID NO 10 or 12,preferably a polypeptide comprising the sequence represented in SEQ IDNO 12, and wherein the double stranded RNA molecule (b) comprises thebinding motif represented in FIG. 41, or as represented in any of SEQ IDNOs 17 to
 34. 15. A method according to claim 6, wherein in (a) the RNAbinding domain is MS2 or a fragment thereof, said fragment comprisingthe RNA binding domain and wherein the double stranded RNA molecule (b)comprises a nucleotide sequence corresponding to the binding motif forbinding to MS2 or a fragment thereof.
 16. A method according to claim15, wherein in (a) the RNA binding domain is a polypeptide comprisingthe sequence represented in SEQ ID NO 14, and wherein the doublestranded RNA molecule (b) comprises the binding motif represented inFIG. 42, or as represented in any of SEQ ID NOs 35 to
 40. 17. A methodaccording to claim 6, wherein said pest species is chosen from the groupcomprising yeast, fungi, insects and nematodes.
 18. A nucleic acidencoding an RNA delivery molecule as defined in claim
 1. 19. A nucleicacid according to claim 18 comprising an in frame signal sequence fordirecting the encoded polypeptide to a plant organel, such as achloroplast, a plastide, a mitochondrium.
 20. A vector comprising anucleic acid of claim
 18. 21. A vector according to claim 20, whereinthe nucleic acid is under the control of regulatory sequences forexpression of the nucleic acid in a plant cell.
 22. A vector accordingto claim 20 further comprising a tissue-specific or cell-specificpromoter.
 23. A vector according to claim 22, wherein the promoter is aninducible or a constitutive promoter.
 24. A host cell comprising anucleic acid according to claim 18, or a vector according to claim 20.25. A host cell according to claim 24, wherein said host cell is abacterial, yeast, fungal or plant cell.
 26. A composition comprising atleast one RNA delivery molecule as defined in claim 1, and optionallyfurther comprising at least one suitable excipient.
 27. A compositioncomprising: a) at least one RNA delivery molecule according to claim 1,and b) at least one RNA molecule which comprises double-stranded RNAcomprising annealed complementary strands, one of which has a nucleotidesequence which is complementary to at least part of a target nucleotidesequence of a target gene of a pest species, and which further comprisesa nucleotide sequence which binds to the RNA-binding domain of the RNAdelivery molecule of (a), and optionally further comprising at least onesuitable excipient.
 28. A kit comprising at least one RNA deliverymolecule as defined in claim
 1. 29. A method for producing an RNAdelivery molecule according to claim 1 comprising: introducing into ahost cell an isolated DNA molecule encoding the RNA delivery moleculeaccording to claim 1; growing the host cell under conditions suitablefor expression of the RNA delivery molecule; and isolating the RNAdelivery molecule produced by the host cell.
 30. A transgenic plantresistant to a pest species, an essential derived variety thereof, plantpart, plant cell or protoplast thereof obtainable by a method accordingto claim
 10. 31. A transgenic plant, essentially derived varietythereof, plant part, plant cell or protoplast thereof which comprises anucleic acid encoding an RNA delivery molecule as defined in claim 1,wherein said nucleic acid is heterologous to the genome of saidtransgenic plant, or an essentially derived variety, plant part, plantcell or plant protoplast thereof.
 32. A plant, essentially derivedvariety thereof, plant part, plant cell or protoplast thereof whereinthe plant, essentially derived variety thereof, plant part, plant cellor protoplast thereof has been transformed with a nucleic acid encodingan RNA delivery molecule as defined in claim
 1. 33. A plant, essentiallyderived variety, plant part, plant cell or protoplast thereof whichco-expresses: a) an RNA delivery molecule according to claim 1, and b)an RNA molecule which comprises double-stranded RNA comprising annealedcomplementary strands, one of which has a nucleotide sequence which iscomplementary to at least part of a target nucleotide sequence of atarget gene in a pest species, and which further comprises a nucleotidesequence which binds to the RNA-binding domain of the RNA deliverymolecule of (a).
 34. The plant according to claim 32, wherein the planthas been stably transformed.
 35. The plant according to claim 32,wherein the plant has been transiently transformed.
 36. A transgenicplant which comprises a vector according to claim
 20. 37. Progeny of aplant or essentially derived variety thereof as claimed in claim 30,obtained in a generative or vegetative manner.
 38. Parts or derivativesof plants obtainable from a plant or essentially derived variety thereofas claimed in claim
 30. 39. The parts or derivatives of plants accordingto claim 38 comprising leaves, stems, roots, shoots, cuttings orexplants and the like, protoplasts, somatic embryos, anthers, petioles,cells in culture, seeds, flowers, fruits and tubers.
 40. A chimeric RNAimolecule comprising at least one nucleotide sequence recognized by anRNA-binding protein or RNA-binding domain, for instance as representedin any of SEQ ID NOs 15 to 40, or an RNA secondary structure asrepresented in FIG. 41 or 42, and, at least one nucleotide sequencecorresponding to a target nucleotide sequence of a target gene of a pestspecies.
 41. A chimeric RNAi molecule according to claim 40, asrepresented in any of SEQ ID NOs 41 to 52 and in any of SEQ ID NOs 54 to56, and in any of SEQ ID NOs 57 to 71, and in any of SEQ ID NOs 72 to86.
 42. A complex comprising an RNA delivery molecule according to claim1, and a chimeric RNAi molecule according to claim
 40. 43. Use of an RNAdelivery molecule according to claim 1 for delivery of a double strandedRNA molecule to a target sequence in a pest species.
 44. Use of an RNAdelivery molecule according to claim 1 for producing a transgenic plant.45. Use of a complex according to claim 42 for down-regulatingexpression of a target gene in a pest species.
 46. Use of a complexaccording to claim 42 for producing a transgenic plant that is resistantto a pest species.